Rodrigo Figueroa, a postdoctoral researcher at Harvard University’s Department of Organismic and Evolutionary Biology, spearheaded this extensive investigation. Motivated by his doctoral work on fossilized ray-finned fish brains, Figueroa identified a glaring gap in neuroscientific research: the paucity of data on living fish brains outside a handful of model species, such as the zebrafish. This gap presented a critical obstacle to deciphering the evolutionary pathways of brain morphology in vertebrates. To address this, Figueroa initiated a large-scale project employing specialized CT scanning to meticulously document the internal cranial structures of 87 ray-finned fish species spanning more than 70 families.

The study’s revelations challenge long-standing assumptions about the relationship between brains and their enclosing skull cavities. Traditionally, it has been presumed, especially among mammals and reptiles, that the brain snugly occupies the internal skull space. Contrarily, the scan data divulged a startling decoupling of brain volume from cranial size in ray-finned fishes. A key metric, the Brain Endocast Coefficient—calculated as the ratio of brain volume to intracranial volume—exhibited extraordinary variability. Most mammals and reptiles display ratios approaching or exceeding 80%, but ray-finned fishes’ ratios typically lie around 40–50%, with some species having brains that occupy less than 5% of their skull cavities.

This deviation suggests that many fish possess large cranial spaces filled with cerebrospinal fluid, blood vessels, or even specialized tissues responsible for immune function and blood production. The presence of such tissues fundamentally alters our interpretation of endocasts—internal molds of skull cavities often used to infer brain morphology, particularly in paleontological contexts. Stephanie Pierce, a senior co-author and curator of vertebrate paleontology at Harvard’s Museum of Comparative Zoology, emphasized that for decades, the scientific community used endocast shapes as proxies for brain structure. However, the data from ray-finned fishes show this assumption can be misleading, underscoring the uncoupled evolutionary trajectories of brains and skulls in these species.

Environmental factors also play a critical role in shaping the brain-to-skull volume ratio observed across species. Statistical analyses within the study link this ratio to the depth at which fish live. Deep-sea species, whether dwelling in the dark ocean floor or the less explored water column, tend to exhibit smaller brains relative to their skull size compared to their shallow-water counterparts. This suggests that the evolutionary pressures of extreme environments, such as the high pressure and low light of the deep sea, could favor anatomical adaptations favoring protection and physiological regulation over brain size.

The protective function of the increased cranial space surrounding tiny brains may act like a biological shock absorber—meningeal tissues enveloping the brain provide a buffer against mechanical impacts or drastic pressure fluctuations. This biological configuration could represent an unrecognized evolutionary strategy for vertebrates living under challenging physical conditions, balancing the demands of neural function with physiological resilience.

Furthermore, the research unveiled intriguing ontogenetic patterns—how brain size relative to skull volume changes throughout an individual’s growth. For example, the bowfin fish exhibits nearly complete skull occupancy by the brain at hatchling stage, which declines sharply to roughly 20–30% in adults. Such drastic changes contrast starkly with more moderate developmental brain-to-skull ratios seen in birds and mammals. In the ancient coelacanth fish, the brain fills almost the entire cranial cavity during youth but shrinks dramatically to a mere 4% of intracranial space in mature individuals, illuminating evolutionary adaptations spanning hundreds of millions of years.

The implications of this study transcend ichthyology, offering a cautionary lens through which to evaluate neuroanatomical inferences based on fossils and extant vertebrates alike. Understanding that endocasts may fail as faithful representations of fish brain morphology necessitates a reevaluation of many historic fossil interpretations, a recalibration that could significantly alter evolutionary narratives involving vertebrate brain development.

Beyond challenging assumptions, the research suggests an astonishing array of neurological strategies underpinning the remarkable diversity and ecological dominance of ray-finned fishes. Since so much neuroscience focuses on a handful of species, this broader perspective urges the scientific community to reconsider the evolutionary flexibility of brain architectures. Do diverse brain morphologies drive ecological success, or do the demands and opportunities of varied environments sculpt brain evolution in these species? This vital question remains open, with this study paving the way for future work seeking to unravel these cause-and-effect relationships.

By harnessing museum collections and cutting-edge imaging, this research represents an important milestone, offering an unprecedented morphological map of fish neurodiversity. The findings clearly underscore that the highly conserved brain-to-skull relationships commonly observed in mammals and birds are the exceptions in the vertebrate lineage, not the standard. Understanding this diversity enriches our knowledge of brain evolution’s vast landscape, expanding concepts of what “normal” brain function and structure may entail.

Figueroa’s ambitious five-year initiative is only the first phase in what promises to be an immense and ongoing endeavor. Scanning 87 species out of tens of thousands is a monumental task, but the insights gained provide a compelling foundation for continued exploration. The study’s comprehensive approach, combining evolutionary biology, neuroscience, and paleontology, exemplifies interdisciplinary scholarship at its finest and opens avenues for understanding vertebrate adaptability and resilience on both micro and macro evolutionary scales.

In summary, this novel research unravels the complex and unconventional relationships between fish brain morphology and cranial anatomy, illuminating how environmental pressures, evolutionary history, and developmental trajectories interplay intricately. It challenges existing paradigms in vertebrate brain study and invites a broader reexamination of how brains evolve within protective structures. This deeper understanding of the ray-finned fishes’ neurological diversity not only reshapes ichthyological knowledge but provides a broader template for comparative neurobiology, with potential ripple effects spanning evolutionary theory, paleobiology, and even biomedical sciences.

Subject of Research: Not applicable
Article Title: The ray-finned fish blackbox: unprecedented morphological diversity and the interplay between brain and endocast.
News Publication Date: 6-May-2026
Web References: http://dx.doi.org/10.1098/rspb.2025.3277
Image Credits: Rodrigo Figueroa (PRSB 2026)
Keywords: ray-finned fishes, brain evolution, neuroanatomy, CT scanning, brain endocast coefficient, vertebrate neuroscience, evolutionary biology, cranial morphology, deep-sea fish, ontogeny, cerebrospinal fluid, comparative neuroanatomy

Cell Painting represents a cutting-edge high-throughput microscopy method that labels multiple cellular components with fluorescent dyes, capturing thousands of images that reveal subtle morphological changes in cells exposed to diverse compounds. According to the study’s corresponding author, Dr. Michael Mancini, professor of molecular and cellular biology and director of Baylor’s Integrated Microscopy Core, this technology allows researchers to observe cellular responses at an unprecedented resolution. By applying custom image analysis pipelines, the team quantified dynamic alterations in cellular structures, providing a detailed phenotypic fingerprint of how each flavonoid interacts with cancer cells.

One of the major challenges of such high-content screening approaches is the sheer volume of data generated. Each Cell Painting experiment can produce over 57,000 confocal microscopy images per plate, a dataset too vast for manual analysis and often requiring substantial computational resources. To overcome this bottleneck, Dr. Mancini’s lab developed SPACe (Swift Phenotypic Analysis of Cells), a novel computational tool capable of individually assessing thousands of cells across numerous experimental plates. Impressively, SPACe can operate efficiently on standard desktop computers, making large-scale drug screening accessible to laboratories regardless of their computational infrastructure.

Applying this powerful methodology, the research team analyzed a library of 244 flavonoid compounds against three widely studied bladder cancer cell lines. Their findings revealed six flavonoids exhibiting significant cytotoxicity, effectively eliminating malignant cells without harming normal bladder cells. Among these were flavopiridol and rotenone, compounds already known for their toxic effects, thereby validating the accuracy of their screening approach. Intriguingly, some flavonoids acted through inducing DNA damage in the cancer cells, while others disrupted mitochondrial function—a critical pathway for cellular energy production—signaling multiple therapeutic mechanisms within this compound class.

Beyond traditional two-dimensional cultures, the study advanced towards more physiologically relevant models, including 3D spheroids and chorioallantoic membrane (CAM) systems, which better mimic tumor architecture and microenvironment. Three of the toxic flavonoids were found to reduce tumor growth in these 3D culture systems as well, reinforcing their potential clinical utility. Significantly, these compounds did not inhibit growth in normal bladder cells, suggesting a degree of cancer cell specificity that could minimize harmful side effects in future therapies.

Among the standout compounds is xanthohumol, a flavonoid derived from hops and found in certain types of beer. The study uncovered that xanthohumol-induced cell death was tightly linked to a reduction in lipid metabolism, particularly a pronounced decrease in the number of lipid droplets within cancer cells. Lipid droplets serve not only as energy stores but also as mediators of cellular signaling and stress responses, marking a novel mechanism of flavonoid-induced cytotoxicity. The possible correlation between xanthohumol consumption and bladder cancer incidence presents a fascinating avenue for epidemiological exploration.

The implications of this research extend well beyond the identification of promising flavonoids. By harnessing the combined power of Cell Painting and SPACe, the Baylor team demonstrated a scalable and precise platform for phenotypic drug discovery that captures the complex heterogeneity of cancer cell populations. This approach allows scientists to classify compounds based on their distinct cellular impact profiles, accelerating the next generation of targeted oncology therapeutics.

Flavonoids themselves are ubiquitously present in fruits, vegetables, and beverages, which raises intriguing possibilities about natural dietary components contributing to cancer prevention or therapy. However, the translation of these in vitro findings to clinical applications requires rigorous validation, including assessment of flavonoid safety, bioavailability, and efficacy in living organisms. The authors emphasize ongoing plans to test these compounds in animal models bearing human bladder tumors and eventually move towards clinical trials to evaluate their therapeutic potential in patients.

The study was a collaborative effort including researchers Jessica Oceguera, Alejandra Rivera Tostado, Christopher D. Candler, Elina Mosa, Kazem Safari, and Maureen G. Mancini. These contributors brought expertise spanning molecular biology, microscopy, and computational analysis, while their institutional support included Baylor College of Medicine and the Texas A&M University’s GCC Center for Advanced Microscopy and Image Informatics.

Funding for this research was provided by multiple prestigious grants, notably from the Cancer Prevention and Research Institute of Texas (CPRIT), the GCC Center for Precision Environmental Health, and the Dan L Duncan Comprehensive Cancer Center. These support mechanisms highlight the critical investment required to facilitate transformative cancer research employing cutting-edge technologies.

As bladder cancer continues to rank as the fifth most common cancer in the United States, causing over 16,000 deaths annually, the need for innovative treatments is urgent. Current clinical practices, while effective at tumor removal and relapse control, often struggle with residual disease that can metastasize. The identification of flavonoids exhibiting selective cytotoxicity against bladder cancer cells offers a hopeful new avenue for improving patient outcomes through less toxic and potentially more effective therapies.

In summary, the marriage of phenotypic screening technologies with natural product libraries exemplified in this study sets a new paradigm in oncology drug discovery. Flavonoid compounds such as xanthohumol exhibit unique cellular interactions that disrupt cancer metabolism and genomic integrity, positioning them as attractive candidates for future therapeutics. This exciting research not only generates a wealth of actionable knowledge but also opens the door to safer, more accessible, and finely tuned cancer treatments, potentially redefining the therapeutic landscape for bladder cancer.

Subject of Research: Human tissue samples

Article Title: A phenotypic screen identifies xanthohumol and other flavonoids as killers of bladder cancer

News Publication Date: 22-Apr-2025

Web References:

• Pharmacological Research – Natural Products Journal
• DOI: 10.1016/j.prenap.2025.100236

Keywords: Human health, Imaging, Microscopy, Organismal biology, Oncology