The study, published in BMC Genomics by a research group led by Xinxin Cao, Lihui Zhang, Kaidong Liu, Jinshan Zhao and Hegang Li, focused on dairy goat embryos at 55 to 65 days of gestation, a critical window during which the gonads commit to their developmental trajectories. The team analyzed three groups of embryos: genetic females, genetic males, and intersex fetuses affected by polled intersex syndrome. Using single-cell RNA sequencing, the researchers profiled the gene expression of thousands of individual cells, then combined this with tissue morphology and immunofluorescence staining to confirm where key proteins were actually located within the developing organs.

Single-cell RNA sequencing works by dissociating a tissue into its component cells and capturing the messenger RNA from each one, allowing researchers to see which genes are active in which cells rather than averaging signals across a whole organ. In this study, that approach revealed striking cellular heterogeneity within the embryonic gonads. The team identified distinct populations of germ cells, the precursors of eggs and sperm, alongside a diverse cast of somatic cells, including stromal cells, granulosa cells, endothelial cells, immune cells, and, in males, Sertoli cells, interstitial cells and epithelial cells. The composition and proportions of these populations differed dramatically between female, male and intersex gonads.

One of the most consequential findings concerns where the different gonadal lineages come from. Through computational reconstruction of differentiation trajectories, a technique known as pseudotime analysis that orders cells along a developmental path based on their gene expression, the researchers found evidence that stromal cells may initiate the development of female and intersex gonads, while epithelial cells appear to serve as the origin of male gonadal cells. This suggests that the cellular ancestry of a gonad may be set remarkably early, and that in intersex embryos the developmental program may be steered toward a male-like trajectory from its starting point.

The molecular culprits behind sex reversal are familiar names in developmental biology, but the study adds new nuance. The researchers documented aberrant expression of SOX9, AMH and DMRT1, a trio of genes that together drive testis formation. SOX9 is the master switch of testis development; AMH, produced by Sertoli cells, triggers the regression of female reproductive structures; and DMRT1 reinforces the male program. In the intersex gonads, these genes were misexpressed in ways consistent with a partial female-to-male conversion. The finding confirms that the same core genetic circuitry implicated in disorders of sex development in humans is also at work in goats with polled intersex syndrome.

Perhaps the most exciting aspect of the study is the identification of new candidate regulators. The researchers flagged PHLDB2, ZNRF3 and TEAD1 as novel genes potentially involved in orchestrating sex reversal. ZNRF3 is known from other contexts to participate in Wnt signaling regulation, a pathway with well-established roles in ovary determination, while TEAD1 sits at the heart of the Hippo signaling pathway, which governs organ size and cell fate. Their emergence from an unbiased single-cell screen suggests that the network controlling gonadal sex determination is even more intricate than the canonical SOX9-centered model implies, and opens new avenues for experimental validation.

The study also delivered a surprise from the immune compartment. The team detected TREM2-positive macrophages within the goat embryonic gonads, a cell population that had not previously been characterized in this setting. Macrophages are best known as immune scavengers, but growing evidence implicates them in shaping tissue environments during organogenesis. The researchers propose that multiple genes expressed in these and other gonadal cells may modulate sex reversal by altering the gonadal microenvironment, the local signaling milieu in which germ cells and somatic cells make their fate decisions. In other words, sex determination may not be a purely cell-autonomous process but one influenced by the surrounding cellular ecosystem.

In a finding that borders on the unexpected, the analysis also implicated circadian rhythm-related genes in early gonadal development and the formation of intersex traits. Circadian genes are classically associated with daily biological rhythms, yet they have increasingly been shown to influence developmental timing in various tissues. Their differential expression across female, male and intersex gonads hints that the internal clock machinery may intersect with the sex determination network, a connection that, if confirmed experimentally, would add an entirely new dimension to how scientists think about disorders of sexual development.

The practical implications for livestock breeding are immediate. Polled intersex syndrome arises in goats bred for the polled, or hornless, trait, because the genetic mutation responsible for hornlessness is tightly linked to the intersex condition. Understanding precisely which cells and genes go awry during early gonad formation could enable breeders to select against the syndrome more effectively, protecting productivity in dairy herds. The single-cell atlas generated by this study provides a molecular reference that could support marker-assisted selection or even gene-editing strategies aimed at decoupling the polled trait from intersex development.

But the study’s ambitions extend well beyond the barnyard. The authors emphasize that polled intersex syndrome in goats serves as a valuable natural model for human reproductive defects, including disorders of sex development that affect roughly one in thousands of newborns worldwide. Because the goat gonad develops through mechanisms broadly comparable to those in humans, the cellular and molecular pathways mapped here, from stromal and epithelial origins to SOX9, AMH and DMRT1 dysregulation, from immune modulation to circadian influences, offer a framework for investigating why and how human sex development sometimes diverges. As single-cell technologies continue to mature, studies like this one demonstrate that even a familiar farm animal can illuminate some of the most fundamental and intimate questions in human biology: how a tiny embryonic organ decides what it will become, and what happens when that decision goes awry.

Subject of Research: Single-cell transcriptomic analysis of intersex gonad initiation and early sex differentiation in dairy goat embryos

Article Title: Single-cell RNA sequencing analysis reveals new insights for intersex gonad initiation and early sex differentiation of dairy goats

Article References: Cao, X., Zhang, L., Liu, K., Zhang, Y., Liu, X., Xu, D., Lin, X., Campo, F. P., Sun, M., Qin, Z., Gao, X., He, J., Zhao, J., & Li, H. (2026). Single-cell RNA sequencing analysis reveals new insights for intersex gonad initiation and early sex differentiation of dairy goats. BMC Genomics. https://doi.org/10.1186/s12864-026-13404-4

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13404-4

Keywords: single-cell RNA sequencing, polled intersex syndrome, dairy goats, sex differentiation, gonad development, SOX9, AMH, DMRT1, pseudotime analysis, germ cells, macrophages, circadian rhythm genes

The research team, led by investigators at the First Affiliated Hospital of Xinjiang Medical University, combined single-cell RNA sequencing, spatial transcriptomics, CRISPR-based gene editing, pharmacological pathway modulation, and xenograft modeling to trace SOX9’s role across both time and tissue space. Using a publicly available single-cell dataset, they reconstructed a pseudotime trajectory with Monocle3, mapping the continuous transition from astrocytes to malignant glioma cells. The analysis revealed substantial cellular heterogeneity within glioma samples, encompassing malignant cells alongside astrocytes, macrophages, T cells, B cells, monocytes, neurons, fibroblasts, and endothelial cells.

The most consequential finding to emerge from the trajectory analysis was SOX9’s temporal behavior. Its expression was concentrated almost exclusively in astrocyte and malignant cell populations, and along the inferred developmental arc it peaked early and then declined steadily as cells matured into malignant states. Correlation analysis confirmed this downward trend, with Pearson and Spearman coefficients both strongly negative and highly significant. At the level of trajectory nodes, SOX9 dominated the early, astrocyte-rich segments and faded in the terminal malignant zones, a pattern inconsistent with the conventional view of SOX9 as a constitutively active stemness factor.

To probe what SOX9 might be doing during that early window, the researchers scored the activity of the Hippo pathway, a master regulator of organ size and stem cell fate whose downstream effectors YAP and TAZ are established drivers of glioblastoma plasticity. Using Gene Set Variation Analysis, they quantified an upstream kinase module, including MST1/2, LATS1/2, SAV1, MOB1A/B, NF2, and WWC1, and a canonical YAP/TAZ target module containing genes such as CTGF, CYR61, ANKRD1, AXL, and BIRC5. Both modules showed inverse relationships with SOX9 expression, and generalized additive modeling revealed that pseudotime and SOX9 each contributed independent, nonlinear effects on pathway activity. In other words, the SOX9–Hippo relationship was phase-dependent, strongest during early-to-intermediate stages of the transition and not a simple monotonic association.

Spatial transcriptomics added a second, geographic dimension to the story. Analyzing four anatomically distinct regions of glioblastoma tissue—the tumor–normal interface, the pure tumor core, the perivascular compartment, and the tumor–necrosis interface—the team mapped SOX9 expression against the probability that each spatial spot contained malignant cells. The coupling between SOX9 and malignancy was weak or unstable at the tumor edges and the necrotic margin, modest in the tumor core, and most robust in the perivascular niche. There, SOX9 expression was markedly elevated, correlations with malignant cell probability were strongest, and neighborhood enrichment and spatial autocorrelation statistics all confirmed significant co-localization.

This regional specificity is biologically meaningful. The perivascular niche has long been recognized as a reservoir for stem-like glioblastoma cells, bathed in vascular, hypoxic, and paracrine signals that nurture cellular plasticity. The findings suggest that SOX9-dependent reprogramming is not only time-restricted but niche-conditioned, with perivascular regions providing the most permissive anatomical context for effective SOX9–Hippo–YAP/TAZ coupling. Elsewhere in the tumor, downstream malignant programs may be sustained through alternative inputs, weakening the spatial coherence of the axis.

Functional experiments brought the correlation studies into the laboratory. Using lentiviral vectors, the team generated U87 glioma cells stably overexpressing SOX9 and used CRISPR/Cas9 to knock out the gene in U251 cells, validating the edits by Sanger sequencing and confirming a frameshift-inducing deletion in the knockout clone. SOX9 overexpression modestly increased proliferation, migration, and invasion while reducing apoptosis, whereas SOX9 knockout produced the opposite phenotype across wound-healing, Transwell invasion, and flow-cytometric apoptosis assays. Critically, treatment with XMU-MP-1, an inhibitor of the upstream Hippo kinases MST1/2, partially rescued the defects caused by SOX9 loss, linking SOX9 function experimentally to Hippo pathway state.

Phosphorylation-level Western blotting sharpened the mechanistic picture. SOX9 overexpression raised the ratio of phosphorylated to total YAP and lowered the phosphorylated-to-total MOB1 ratio, while SOX9 knockout produced the reciprocal pattern. XMU-MP-1 shifted both readouts toward the SOX9-overexpression signature, and total MOB1 remained unchanged across groups, indicating that the pathway rewiring was phosphorylation-dependent rather than a simple change in protein abundance. YAP and TAZ mRNA and protein levels rose with SOX9 gain and fell with SOX9 loss, and drug treatment partially restored them in SOX9-deficient cells, extending the transcriptomic associations to protein-level pathway readouts.

In vivo, the story held. Subcutaneous xenografts in nude mice showed that SOX9 overexpression significantly accelerated U87-derived tumor growth from day 14 onward, while SOX9 knockout markedly suppressed U251-derived tumors. XMU-MP-1 treatment further enlarged SOX9-overexpressing tumors and partially reversed the growth inhibition caused by SOX9 loss. Histopathology mirrored these dynamics: SOX9-overexpressing tumors displayed increased necrosis and nuclear atypia, whereas knockout tumors showed milder pathology, and the drug partially reversed both patterns. Ki67 immunohistochemistry confirmed the corresponding changes in proliferative activity, and CD68 staining revealed that myeloid and macrophage-like cell accumulation also shifted with SOX9 status, adding an immune dimension to the tumor microenvironmental effects.

Taken together, the study proposes what the authors call an early priming–late decoupling model. SOX9 acts early, at the moment of astrocyte-to-malignant conversion, to initiate Hippo–YAP/TAZ-linked malignant reprogramming. Once downstream transcriptional networks consolidate, the tumor becomes progressively less dependent on sustained SOX9 expression, which explains why stemness programs can persist in advanced disease even as SOX9 levels fall. The translational implication is pointed: therapies aimed at SOX9 may work best before malignant programs fully consolidate, whereas in later-stage tumors, blocking SOX9 alone may prove insufficient. The results argue for stage-specific and niche-aware strategies targeting the SOX9/Hippo/YAP–TAZ axis, particularly in the perivascular compartment where the axis is most strongly engaged. Limitations remain, including the reliance on public cohorts of limited size, established cell lines, and subcutaneous rather than orthotopic models, and the absence of YAP/TAZ nuclear localization data or a second pathway inhibitor. Even so, the study repositions SOX9 from a static stemness marker to a dynamic state-switch regulator, and it provides a conceptual framework for timing future interventions against one of medicine’s most stubborn cancers.

Subject of Research: The temporal role of SOX9 in priming Hippo–YAP/TAZ signaling during glioblastoma stemness acquisition

Article Title: Early SOX9 Activation Primes Hippo–YAP/TAZ Rewiring During Glioblastoma Stemness Acquisition

Article References: Early SOX9 Activation Primes Hippo–YAP/TAZ Rewiring During Glioblastoma Stemness Acquisition. (n.d.). https://doi.org/10.1111/jcmm.71341

Image Credits: AI Generated

DOI: 10.1111/jcmm.71341

Keywords: glioblastoma, SOX9, Hippo pathway, YAP/TAZ, glioma stem cells, pseudotime analysis, spatial transcriptomics, perivascular niche, XMU-MP-1, CRISPR, tumor plasticity, xenograft