The study, published in BMC Genomics, focused on N6-methyladenosine, commonly abbreviated as m6A. This modification is the most abundant internal chemical tag found on messenger RNA in eukaryotic cells. Rather than altering the genetic code itself, m6A acts like a set of sticky notes on the transcript, influencing how efficiently each message is read, how stable it remains, and how it is processed. Scientists have known for over a decade that m6A shapes development in organisms ranging from plants to mammals, but its role in the skin biology of livestock has remained largely uncharted territory.

To probe this frontier, the research team, led by Binpeng Xi and Jianbin Liu of the Xinjiang Academy of Agricultural and Reclamation Science, employed two complementary sequencing technologies. The first, MeRIP-seq, uses antibodies that specifically recognize the m6A mark, allowing researchers to immunoprecipitate methylated RNA fragments and map exactly where on the transcriptome these tags reside. The second, RNA-seq, measures the abundance of every transcript, revealing which genes are switched up or down. By running both methods on skin samples from three newborn black-fleeced lambs and three 179-day-old white-fleeced lambs, the team could compare not only gene expression but also the epitranscriptomic landscape that governs it.

The mapping results revealed a strikingly consistent architecture of methylation across both age groups. Most m6A peaks clustered within coding sequences and near stop codons, the regions where the cellular machinery translates RNA into protein. The team also detected the canonical GGACU motif, the sequence signature most commonly recognized by m6A-binding proteins, in both the newborn and older animals. This consistency suggests that the fundamental machinery of RNA methylation operates similarly at both stages of coat development, while the differences lie in which specific genes carry the marks.

Those differences proved substantial. Across three paired comparisons between individual lambs of the two age groups, the researchers identified between 1,273 and 1,716 differential m6A peaks, alongside 400 to 1,657 differentially expressed genes. Functional enrichment analysis of these changing genes and peaks highlighted biological processes intimately tied to the coat transition: hair follicle development, epidermal morphogenesis, extracellular matrix remodeling, and focal adhesion. Signaling pathways long implicated in pigmentation and skin renewal also featured prominently, including Wnt, TGF-beta, PI3K-Akt, Notch, and melanogenesis-related cascades. The convergence of methylation changes and expression shifts on these pathways provides compelling circumstantial evidence that RNA modification participates in reprogramming the skin as fleece color flips.

One of the study’s most technically interesting contributions is its integrative analysis of how methylation status and transcript abundance couple. The researchers observed distinct coupling patterns depending on the comparison. In one pairing, hypermethylation coincided with downregulated expression, a pattern consistent with m6A accelerating transcript decay. In the other two comparisons, the predominant pattern was the opposite: hypomethylation paired with increased expression, suggesting that removal of the mark stabilizes certain messages during the white-fleece phase. This comparison-dependent coupling underscores a key principle of epitranscriptomics: the functional consequence of an m6A mark depends on its position within the transcript and the cellular context in which it appears.

From this integrated dataset, the team prioritized a shortlist of candidate m6A-associated genes that may drive the color transition. The list includes FZD7, a receptor in the Wnt signaling pathway with established roles in hair follicle cycling; GAS1, a growth arrest factor involved in developmental signaling; COL4A1, a structural collagen of the basement membrane; PXDN, an enzyme involved in collagen cross-linking; LAMC3, a laminin chain component of the extracellular matrix; ARID1A, a chromatin remodeling factor; FA2H, an enzyme in fatty acid metabolism; and RAPGEFL1, a signaling scaffold. The presence of extracellular matrix and Wnt pathway genes on this list fits the biology of the transition, since shifting from black to white fleece requires not only changes in pigment production by melanocytes but also structural remodeling of the follicle environment in which those pigment cells operate.

To validate their sequencing results, the researchers performed quantitative real-time PCR on selected genes, confirming that the expression trends observed by RNA-seq held up under an independent measurement method. This verification step strengthens confidence in the dataset, though the authors are careful to note that the m6A sites themselves have not yet been individually validated by orthogonal techniques, and functional studies of the candidate genes remain to be done. The work is explicitly framed as a candidate-generation study, laying the groundwork for targeted follow-up experiments rather than closing the case.

The authors also flag an important caveat: in their design, age and coat-color stage are confounded, since every older lamb was both white-fleeced and 179 days old. The molecular differences they detected therefore reflect the developmental transition as a whole, and disentangling which changes are specifically responsible for pigment switching will require additional experiments, potentially including time-course sampling at intermediate ages or comparisons with sheep breeds that do not undergo a color change. This honest framing is a model of scientific caution in a field where epitranscriptomic datasets can invite overinterpretation.

Nevertheless, the implications extend well beyond a single sheep breed. Coat color is an economically significant trait in wool and meat production, and understanding its developmental regulation could inform breeding strategies. More broadly, the study adds livestock skin biology to the growing map of tissues where m6A shapes developmental programs, joining work on embryogenesis, neurodevelopment, and immune differentiation. As the tools for precise m6A site validation and genome editing mature, the candidate genes identified here offer a concrete starting point for functional dissection. The humble Junken lamb, transforming from black to white in its first six months of life, has now given scientists a molecular thread to pull, and the fabric it unravels may reshape how we think about the epigenetic control of visible traits in farm animals.

Subject of Research: m6A RNA modification and gene expression during developmental coat color transition in Junken meat sheep

Article Title: Integrated MeRIP-seq and RNA-seq analyses identify candidate m6A-associated genes during developmental coat color transition in Junken meat sheep

Article References: Xi, B., Zhao, S., Yu, Q., Zhou, H., Zhang, W., Chen, Y., Cheng, R., Wang, Z., Yang, H., & Liu, J. (2026). Integrated MeRIP-seq and RNA-seq analyses identify candidate m6A-associated genes during developmental coat color transition in Junken meat sheep. BMC Genomics. https://doi.org/10.1186/s12864-026-13311-8

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13311-8

Keywords: m6A methylation, MeRIP-seq, RNA-seq, coat color transition, Junken meat sheep, hair follicle development, melanogenesis, Wnt signaling, epitranscriptomics, skin biology, livestock genetics, BMC Genomics

Hair follicles are among the smallest and most numerous organs in the human body, yet their formation follows many of the same fundamental principles that govern the development of larger and more complex organs. Cells must first gather in the correct location, establish a precise orientation, communicate with neighboring cells and then specialize into distinct populations with different jobs. Those populations must continue to grow and reorganize until they produce a functional structure. Because mouse skin contains hundreds of follicles progressing through different developmental stages at the same time, it provides a natural collection of biological “frames” that can be arranged to reconstruct development over time.

“Our four-dimensional map of the hair follicle from mice serves as a model system for understanding broad-stroke fundamentals of how organs develop,” says Reza Kalhor, an associate professor of biomedical engineering at the Johns Hopkins University School of Medicine, who led the study. The researchers describe their approach as four-dimensional because it combines three-dimensional molecular information with a fourth dimension inferred from the developmental age of each follicle. Rather than watching one follicle continuously, they compare many follicles captured at different stages and use their molecular profiles to place them in chronological order.

The core of the project is a new molecular imaging method called 3D DNase-Enhanced Expression Profiling, or 3DEEP. The technique is designed to overcome a major obstacle in spatial gene-expression analysis: the presence of genomic DNA in tissue. DNA can interfere with chemical reactions used to detect messenger RNA, the short-lived molecules that carry instructions from genes to the cellular machinery responsible for making proteins. Messenger RNA reveals which genes are active in each cell, while its location shows where those cellular programs are operating within the developing organ. By removing genomic DNA from relatively large pieces of skin, the researchers were able to preserve and analyze fragile RNA molecules across entire hair follicles rather than examining only thin slices.

After preparing the tissue, the team labeled millions of RNA molecules according to their positions. The resulting data produced a detailed three-dimensional map of gene activity throughout the skin samples. The researchers then used molecular signatures to classify the different cell types present in each follicle, including cells that form the structural components of the organ and cells that guide its growth and specialization. This combination of spatial position, gene activity and cell identity allowed them to estimate the molecular age of individual follicles. The follicles could then be ordered from their earliest developmental stages to their most mature forms, creating a reconstructed sequence of organ formation.

The maps revealed a tightly coordinated progression. At first, precursor cells formed a localized thickening in the skin. These cells then organized themselves along an axis oriented perpendicular to the skin surface, establishing the basic geometry of the future follicle. In the next phase, the precursor population began differentiating into multiple cell types, each assigned a distinct role in building the organ. Finally, the newly specialized cells expanded, moved and reshaped the developing structure until it became a deep, mature follicle capable of producing a hair shaft. The animation interface created by biomedical engineer Jean Fan allows researchers to explore these changes interactively and examine how cellular patterns shift through the reconstructed timeline.

The scientists also used 3DEEP to compare normal mice with hairless mice carrying a mutation in Foxn1, a gene known to be essential for hair growth. The comparison showed that the mutant follicles did not simply stop growing at the beginning of development. Instead, they entered a delayed and disorganized developmental trajectory. Cells in the follicles of hairless mice were capable of proliferating, or dividing, at a higher rate than cells in normal follicles. However, the mutant cells were less able to mature and adopt specialized identities at the appropriate time. In a developing organ, rapid cell division without correctly timed differentiation can be destructive rather than beneficial.

The researchers say that the Foxn1 mutation disrupted the communication and timing required to coordinate follicle formation. As a result, the developing structures became unstable and collapsed before they could produce hair. “The Foxn1 mutation led to a breakdown in cellular communication and timing, causing the hair follicle organs to structurally collapse before hair had the chance to form,” says Soichiro Asami, the study’s first author and a doctoral candidate in Kalhor’s laboratory. Luis Garza, a professor of dermatology at Johns Hopkins and a co-author, says the maps provide a window into organogenesis by showing how a follicle grows from a small skin thickening into a deep, mature structure.

The researchers believe the method could eventually be applied to a broad range of biological problems. A three-dimensional molecular record of organ development may help scientists determine how inherited mutations alter cellular communication, identify the earliest stages at which developmental disorders emerge and clarify how tumors change their surrounding tissue over time. In hair biology, the same approach could help distinguish whether a condition is caused by defective cell proliferation, failed differentiation, disrupted signaling or a breakdown in the physical architecture of the follicle. Garza says that applying the technology to human samples could ultimately allow researchers to extract far more information from an individual patient and support more personalized treatment strategies.

For now, the study remains a mouse-based demonstration, and the reconstructed time dimension is inferred rather than recorded by continuously observing living follicles. Even so, the ability to capture large tissue regions while retaining molecular and spatial detail marks a significant advance in developmental biology. Kalhor, Asami, Fan, Garza and their colleagues say that 3DEEP offers a way to study organ formation as a coordinated process involving thousands of cells rather than as a series of isolated snapshots. By revealing how location, gene activity, cell identity and developmental timing interact, the technology may provide a new framework for understanding how organs are built—and how seemingly small errors can cause them to fail.

Subject of Research: Three-dimensional and four-dimensional molecular mapping of hair follicle development, including the effects of the Foxn1 mutation in hairless mice.

Web References: Cell research article; CellCarto-3DEEP interactive interface; Reza Kalhor profile; Luis Garza profile.

References: Cell; Johns Hopkins Medicine; National Institutes of Health; Simons Foundation; David and Lucile Packard Foundation.

Keywords: hair follicle development, organogenesis, 3DEEP, spatial gene expression, molecular imaging, four-dimensional biology, Foxn1, hair loss, biomedical engineering, developmental biology, single-cell analysis, tissue mapping