The human body, like those of most multicellular organisms, exhibits numerous instances of suboptimal design. These imperfections arise from evolutionary processes that balance competing demands, often prioritizing immediate reproductive success over long-term well-being and efficiency. As a result, many biological structures and functions are prone to errors, which tend to accumulate and manifest more prominently with age.
These inherent imperfections have driven the evolution of additional layers of complexity aimed at mitigating potential failures. Such complexity would likely be unnecessary if these biological systems had been optimally designed from the outset. Therefore, the presence of intricate mechanisms to counteract inherent errors serves as compelling evidence for evolution and challenges the notion of intelligent design. Examples of these compensatory complexities are abundant across all multicellular organisms.
A pertinent example involves the regulation of transposable elements (TEs), often referred to as "jumping genes." These DNA sequences can move within the genome, potentially causing significant disruptions if not properly controlled. In healthy cells, TEs are kept in check within heterochromatin — a tightly packed form of DNA that serves as a "prison" for these elements. Recent research led by Professor Anjana Rao, Ph.D., at the La Jolla Institute for Immunology, published in Nature Structural & Molecular Biology, has shed light on this control mechanism. The study reveals that the enzyme O-GlcNAc transferase (OGT) plays a crucial role in suppressing TE activity by restraining TET enzymes, thereby maintaining genomic stability.
This intricate system of checks and balances underscores the evolutionary arms race within our genomes, highlighting the complexity that arises from natural selection's ongoing efforts to mitigate the potential harms posed by transposable elements.
What may present a fascinating puzzle for science often deals a fatal blow to creationism — if only its adherents would acknowledge it. However, creationism remains a "brain-dead zombie", artificially kept alive by the manoeuvres of creationist leaders whose power and income rely upon it.
For instance, the recent discovery in China of stone tools exhibiting 'Quina technology', typically associated with Neanderthals, raises intriguing questions for archaeologists and anthropologists. Neanderthals were previously thought to have inhabited primarily western Eurasia, yet these Chinese artefacts, dated to between 50,000 and 60,000 years ago, suggest their influence or presence extended much farther east than previously known. These findings pose fascinating questions regarding ancient human migration and technological exchange.
However, these same discoveries directly contradict creationist beliefs that the Earth is merely 6,000 to 10,000 years old and that humans appeared through a special creation without ancestral links. While science thrives on unanswered questions and continuously adapts its theories based on new evidence, creationism relies rigidly on dogma. When its foundational claims are refuted, the entire belief system crumbles. Religion insists upon unreasonable certainty, whereas science flourishes through reasonable uncertainty.
The discovery of this evidence of Quina Technology was made at the Longtan archaeological site in southwest China by an international group of archaeologists, which included Professor Ben Marwick of Washington University, USA. It is first such discovery in Asia of a technology known to have existed in Middle Palaeolothic Europe and associated there with Neanderthals.
The question is, does this show that Neanderthals were more widespread than we thought, or has their technology been shared with other hominins such as the Denisovans? Or did the same technology arise independently in China?
A major problem for creationists who cling to the delusion that Earth was magically created only 6,000 to 10,000 years ago is that this timescale leaves approximately 99.9975% of Earth's actual history unaccounted for. Consequently, there is an overwhelming abundance of evidence contradicting their beliefs. To maintain their position, creationists are forced to rely on increasingly elaborate mental gymnastics to dismiss the clear indications of an ancient Earth within an even older Universe—evidence consistently revealed and verified by multiple scientific disciplines, including palaeontology and geology.
This is approximately 40,000 years before young Earth creationists claim their proposed deity created a small, flat Earth beneath a dome in the Middle East, along with a man formed from dust and a woman from his rib as the founding couple of the human species.
As with approximately 99.9975% of Earth's history, the vast majority of human history occurred long before the supposed ‘Creation Week’. The established record of human origins and development differs so radically from the narratives found in the Bible and Qur’an that it is remarkable anyone still considers those texts to be authoritative accounts of history or science—or even credible allegories or metaphors for anything resembling reality.
Genetic evidence further shows that, during their migrations, the ancestors of modern Papuans interbred with now-extinct archaic humans known as Denisovans. As a result, while modern Eurasians typically carry around 2% Neanderthal DNA, many populations in Island Southeast Asia and Oceania—including Austronesian peoples—carry up to 3% Denisovan DNA.
The authors, Professor Dylan Gaffney and Marlin Tolla have published an account of the research that went into their book, open access in the online magazine, The Conversation Their article is reproduced here under a Creative Commons License, reformatted for stylistic consistency. The original can be read here.
Autoimmune conditions and allergies provide strong evidence for evolution over intelligent design by highlighting the imperfections and trade-offs inherent in the immune system. These disorders demonstrate how a system shaped by natural selection can prioritize short-term survival at the expense of long-term health, leading to vulnerabilities that are difficult to reconcile with the concept of a perfect designer.
Autoimmune diseases occur when the immune system mistakenly attacks the body’s own tissues. Examples include rheumatoid arthritis, lupus, multiple sclerosis, and type 1 diabetes.
The immune system must strike a delicate balance: it must be reactive enough to fight infections but tolerant enough to avoid attacking the body’s own cells. Evolution has shaped this balance, but it is imperfect. A hyperactive immune system, while better at combating infections, increases the risk of autoimmune diseases.
Now it's beginning to look like we must include the fatal lung disease known as idiopathic pulmonary fibrosis (IPF) to the long list of autoimmune conditions that stem directly from the facts that the immune system evolved and was not intelligently designed.
The very existence of the immune system should prompt creationists to reconsider several basic beliefs and especially claims made by leading proponents, such as Michael J. Behe. Behe suggests that pathogens and the diseases they cause result from 'genetic entropy', a degradation enabled by biblical 'Sin', which supposedly causes genomes to 'devolve', thus creating parasites and pathogens. Other creationists suggest an alternative viewpoint, attributing the existence of parasites and diseases to an evil designer, such as Satan - a claim which is regarded as blasphemous by fundamentalists for whom it is doctrine that there is only one creative entity - God.
In contrast, William A. Dembski argues that any 'complex specified information' (CSI) within the genome must originate from an intelligent designer, typically inferred — though seldom explicitly acknowledged by prominent creationists — as the God described in the Bible or the Qur'an. The genes that enable parasites to evade our immune defences are clear examples of what Dembski would term CSI, whereas Behe regards these genes as 'devolved' from an initially perfect creation.
However, neither Behe nor Dembski adequately addresses the question of who or what designed the immune system itself. Was it the same designer responsible for Dembski's complex specified information, or was it Behe's designer of an initial, perfect creation? If we consider Dembski's argument, it raises a critical question: where is the intelligence in designing an immune system to protect organisms against pathogens created by the very same designer? Regarding Behe's perspective, if the immune system were part of the initial perfect design, why would the designer anticipate 'The Fall' and its consequences unless it was intentionally planned? Alternatively, did all organisms possessing immune defences receive an upgrade after 'The Fall', indicating a supposedly omniscient deity initially failed to foresee the need for such protection? This then raises the question, is the designer either not omniscient or not competent, or did it plan for the 'Fall' and the suffering caused by parasites all along?
Not only are there these gaping flaws in creationism's attempts to account for the immune system within their own theology, including its failure to protect us and its propensity to attack us because the delicate balance referred to above is not robust enough or sensitive enough. The fact is that a perfectly designed immune system should make much of medical science redundant. However, the evidence continues to accumulate that the immune system, like the rest of biology is not the result of intelligent design but of an evolutionary process with all its inherent faults, constraints and inevitable suboptimal compromises.
Evidence strongly supporting the theory that IPF is the result of an autoimmune response by an over-sensitive immune system has been provided by a team of researchers led by Rutgers University, New Jersey, USA. Their findings are being published in the European Respiratory Journal and are described in Rutgers Today:
Evolved macroscopic "snowflake" yeast from the MuLTEE experiment. The large size of the nuclei (yellow) and cells (cyan) are results of whole-genome duplication and aneuploidy.
Credit: Ratcliff Lab
It is a common claim in creationist circles, despite clear evidence to the contrary, that information theory prevents the creation of new genetic information. They argue incorrectly that Shannon Information Theory dictates that the total amount of information in the universe is fixed. According to this flawed view, creating new genetic information would violate the First Law of Thermodynamics, which states that energy can neither be created nor destroyed.
However, this interpretation demonstrates a misunderstanding of both thermodynamics and Shannon Information Theory, as well as how these concepts relate to genetic information. In reality, the creation of new genetic information can be readily observed each time cells replicate, as the total genetic content effectively doubles, The elements the 'information' is composed of are neither created nor destroyed in the process and, as the result of chemical processes, there is less energy in the system, so the laws of thermodynamics are conserved.
Gene duplication and entire genome duplication (polyploidy) are common occurrences in biology, particularly within the plant kingdom, where tetraploidy — possessing twice the usual diploid number of chromosomes — frequently arises. It is also sometime seen in arthropods, amphibians and reptiles.
Tetraploidy often appears spontaneously in laboratory populations of various organisms. Typically, without selective pressures favouring polyploid states, these conditions tend to revert to diploidy after several generations. However, recent studies by scientists at Georgia Tech, conducting multicellular long-term evolution (MuLTEE) research with 'snowflake yeast', Saccharomyces cerevisiae, have demonstrated that under specific selective pressures, polyploidy can become stable and confer advantageous survival traits to the organism.
The selection pressure in this case was selecting the largest yeast cells from which to produce the next generation. The researchers discovered that polyploidy had arisen early on in the experiment, after about 10 generations, and polyploid cells tended to be the largest cells, so a polyploid strain quickly arose and remained polyploid over thousands of generations - far longer than would be expected if selection had been random or unrelated to cell size.
Microscopic image of a section of an electroporated, genetically modified chimpanzee brain organoid. Cell nuclei in blue, precursor cells in magenta, electroporated, genetically modified cells in green and dividing cells in orange.
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Homo habilis is an extinct hominid species that lived between 2.8 and 1.5 million years ago. It is considered to be one of the earliest members of the genus Homo, and its name means "handy man" in Latin, reflecting its ability to make and use tools.
Creationists often struggle to explain why closely related species share identical genes located at the same locus on the same chromosome, typically resorting to the argument of common design rather than common descent. Even more challenging for creationists is when these shared genes exhibit slight modifications that result in significant differences between species, strongly supporting descent with modification.
A compelling recent example involves two genes, NBPF14 and NOTCH2NLB, identified by researchers from the German Primate Center - Leibniz Institute for Primate Research (DPZ) and the Max Planck Institute of Molecular Cell Biology and Genetics. These genes, modified specifically in humans, appear to explain the larger and more complex human brain compared to chimpanzees and bonobos. The research shows that NBPF14 and NOTCH2NLB act synergistically: one gene increases the production of neural progenitor cells, while the other facilitates their transformation into neurons capable of forming more extensive neural connections.
Together, these genetic modifications account for the remarkable increase in the size and complexity of the human brain relative to our closest primate relatives.
For an explanation of how two mutations with a low probability can quickly spread through the gene pool when they act synergistically, see my book Twenty Reasons To Reject Creationism: Understanding Evolution, pages 17-20, in which, using the example of a bacterium and two beneficial mutations acting synergistically, I show that the time take for 50% of the population to have both mutations is actually shorter than the time for 50% of the population to have just one mutation because, the accelerating effect of synergy increases the probability of both being inherited together.
This can explain why a large-brained archaic hominin appeared relatively suddenly in the fossil record. As we shall see, the fact that at least one of our ancestral species went through a narrow genetic bottleneck was ultimately highly beneficial because this reduces the time taken for the whole gene pool to acquire a neutral mutation by genetic drift alone.
Impression of molten Earth at 300 million years old
AI-Generated image (Chat GPT4.5)
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Artistic view of Earth’s interior during mantle solidification in the first hundreds of millions of years of Earth’s history. Gravitational segregation of dense, iron-rich magma (in orange) likely formed a basal magma ocean atop the core, that can explain the present-day structure of the lower mantle.
According to creationist mythology, approximately 6,000 to 10,000 years ago, a supernatural entity conjured Earth into existence out of nothing. This planet, according to the myth, was initially a small, flat surface covered by a dome, centred around the vicinity of the Middle East.
In stark contrast to this mythological narrative, scientific evidence reveals a completely different history — one so profoundly distinct that any metaphorical interpretation of the myth quickly collapses under scrutiny. The Earth is neither flat nor limited to a small region around the ancient Canaanite hills; nor does it possess a dome overhead. No amount of linguistic gymnastics can transform that ancient depiction into anything remotely resembling Earth, although it may well have resembled the perception of Earth from the point of view of scientifically illiterate Bronze Age pastoralists who had never travelled more than a day or two's walk from their homeland and who though the sky was blue because of all the water above the transparent dome overhead.
As though any more refutation of the idea that the Bible contains an inerrant account of actual history, a recent study by a research team led by Assistant Professor Charles-Édouard Boukaré from the Department of Physics and Astronomy at York University in Toronto, Canada, sheds new light on Earth's early geological development. Their research demonstrates how Earth gradually cooled over its first 100 million years, providing valuable insights into the planet’s current thermochemical internal structure.
(C–F) Expanded views of the interaction interface between STAT2 CCD and IRF9 IAD for mouse (C), human (D), Hypanus sabinus (E), and Stegostoma tigrinum (F). The interactions are observed in the crystal structure of the mouse STAT2-IRF9 complex (PDB ID: 5OEN) [19]. For humans and the two cartilaginous fishes, the interactions are based on the modeled structures of the STAT2-IRF9 complex. The key residues involved in the interface are labeled. The phenylalanine (F) on the STAT2 protein is colored in green. The four residues forming the cleft on the IRF9 protein are colored in magenta. The corresponding sequences of the interface area and other details are found in Supporting Information S1: Figure S3.
Recent research conducted by undergraduate students at the University of Nebraska–Lincoln has provided compelling insights into the evolutionary development of the human immune system. Under the guidance of Professor Luwen Zhang, students Vanessa Hubing, Avery Marquis, and Chanasei Ziemann co-authored two significant studies published in the Journal of Medical Virology. Their work elucidates the progression of immune regulatory mechanisms in vertebrates, highlighting the transition to more complex systems with the evolution of jaws. Additionally, they explored how a pseudogene, potentially introduced into primate DNA via a retrovirus approximately 60 million years ago, may have enhanced ancestral immune responses.
These findings offer robust evidence supporting the theory of evolution by demonstrating the gradual and adaptive changes in genetic material that have led to sophisticated immune functions in humans. The identification of a pseudogene's integration into primate DNA and its subsequent role in immunity exemplifies natural selection's influence on genetic composition over millions of years. Such evidence challenges creationist perspectives by providing concrete examples of evolutionary processes shaping complex biological systems, underscoring the dynamic nature of genetic evolution in response to environmental pressures.
During the course of evolution, these factors have evolved as additional layers of complexity to improve and refine a system which, as the product of an unplanned, utilitarian evolutionary process was a suboptimal compromise between the tendencies to over-react to some infections and fail to respond to others. An intelligently-designed sytem would need no such regulatory mechanisms. This is how we can tell that such overly-complex systems were not intelligently designed.
Like all organisms, and particularly pathogenic parasites that colonise our intestines, Clostridioides difficile (C. diff) must compete with other organisms for nutrients. This competition inevitably fuels evolutionary arms races.
For devotees of creationism’s ‘intelligent designer’, C. diff might appear to be a cunning response to medical science's successful use of antibiotics against bacterial pathogens. This is because C. diff is equipped with multiple antibiotic-resistance genes, allowing it to thrive in hospital environments. It often infects patients who are already vulnerable due to other health conditions or compromised immunity, making it a significant medical challenge.
Furthermore, if one follows William A. Dembski's reasoning, the ‘complex specified information’ in C. diff’s genome, which grants it a competitive edge, must logically be attributed to an intelligent designer. Michael J. Behe’s attempt to absolve his version of an intelligent designer by blaming ‘sin’, ‘genetic entropy’, or alleged ‘devolution’ fails here. A mutation that clearly provides an adaptive advantage cannot logically be termed a ‘devolution’ from a supposedly more ‘perfect’ ancestral state.
If creationism’s intelligent designer intended to kick people when they were down, it could hardly have done better than designing C. diff.
How C. diff competes for resource in our gut by waging chemical warfare against the other gut biota is the subject of a paper in the journal Cell Host & Microbe by researchers at Vanderbilt University Medical Center (VUMC). Their findings are described in VUMC News:
Tell me all about Clostridium difficile including its evolutionary origins and pathology in humans.
Clostridium difficile(now officially known as Clostridioides difficile) is a Gram-positive, anaerobic, spore-forming bacterium responsible for a range of gastrointestinal diseases collectively known as Clostridioides difficile infection (CDI). It's particularly notable as a healthcare-associated pathogen, often associated with antibiotic use.
Here's a detailed overview covering taxonomy, evolutionary origins, pathology, epidemiology, and treatment: Taxonomy and classification
Kingdom: Bacteria
Phylum:Bacillota (formerly Firmicutes)
Class:Clostridia
Order:Eubacteriales
Family:Peptostreptococcaceae
Genus:Clostridioides
Species:Clostridioides difficile (formerly known as Clostridium difficile)
The reclassification from Clostridium to Clostridioides reflects genomic analyses showing distinct evolutionary divergence.
Evolutionary origins
Clostridioides difficile diverged from other members of the Clostridium genus millions of years ago, adapting uniquely to colonise and survive in the mammalian gastrointestinal tract.
Genomic studies suggest it has undergone significant horizontal gene transfer, acquiring antibiotic resistance genes and virulence factors, enabling rapid adaptation to clinical environments.
The evolutionary success of C. difficile has been significantly influenced by human activities, particularly widespread antibiotic use, which disrupts normal gut microbiota and creates an ecological niche favouring its growth and pathogenicity.
Microbiology and transmission
It is an obligate anaerobe, thriving without oxygen.
Forms resilient spores that can survive harsh environmental conditions, resisting heat, disinfectants, and drying.
Transmission is mainly faecal-oral, commonly via contaminated surfaces, healthcare workers’ hands, or medical equipment.
Often colonises the human gut asymptomatically, especially in infants, though it can turn pathogenic under conditions of microbiota imbalance (dysbiosis).
Pathogenesis in humans
C. difficile pathogenesis is primarily driven by its two major toxins:
Toxin A (TcdA): an enterotoxin disrupting intestinal cell integrity, causing fluid secretion, inflammation, and diarrhoea.
Toxin B (TcdB): a potent cytotoxin causing significant cellular damage and inflammation.
A third toxin, the binary toxin CDT, found in some strains (e.g., epidemic ribotype 027), may enhance virulence but is less consistently expressed.
Disease progression:
Antibiotic treatment disrupts normal gut microbiota.
Spores of C. difficile germinate into vegetative forms, multiply, and colonise the gut.
Toxins damage colonic epithelium, trigger inflammatory responses, and lead to disease manifestation.
Clinical manifestations
In humans, the clinical spectrum ranges from mild diarrhoea to severe and life-threatening colitis:
Mild to moderate CDI:Watery diarrhoea, abdominal cramping, fever, loss of appetite, nausea.
Severe CDI:Profuse diarrhoea, dehydration, severe abdominal pain, pseudomembranous colitis (characteristic formation of pseudomembranes on the colon mucosa), toxic megacolon, bowel perforation, and sepsis, potentially fatal if untreated.
Risk factors include antibiotic exposure, advanced age, prolonged hospital stays, immunocompromise, proton-pump inhibitor usage, and inflammatory bowel disease.
Epidemiology and public health impact
CDI is primarily hospital -or healthcare-associated, although community-acquired infections have been increasingly documented.
Epidemic strains (e.g., ribotype 027, also known as NAP1 or BI) emerged in the early 2000s, demonstrating increased virulence, toxin production, antibiotic resistance, and morbidity/mortality rates.
Global incidence has been rising, influenced by antibiotic prescription practices and ageing populations, prompting greater vigilance in infection control.
Strict hand hygiene (soap and water preferred over alcohol rub due to spore resistance).
Environmental cleaning with sporicidal disinfectants (e.g., chlorine-based agents).
Prevention and ongoing research
Improved antibiotic stewardship to reduce CDI risk.
Ongoing vaccine development aimed at toxin neutralisation.
Research continues into probiotic formulations and other microbiota-directed therapies for prevention and treatment.
Summary
Clostridioides difficile is a rapidly evolving bacterium whose success is closely tied to human interventions, especially antibiotic use.
Pathogenicity stems from toxin production causing severe inflammation and colonic injury.
CDI remains a significant public health concern globally, demanding continued attention to prevention, effective treatment protocols, and infection control measures.
Understanding its evolutionary dynamics, microbiology, and host interactions is critical in managing and mitigating its clinical impact.
C. diff uses toxic compound to fuel growth advantageThe findings of a new research study increase understanding of the molecular drivers of C. diff infection and point to novel therapeutic strategies aimed at a pathogen that causes about half a million infections in the U.S. each year.
The pathogen C. diff — the most common cause of health care-associated infectious diarrhea — can use a compound that kills the human gut’s resident microbes to survive and grow, giving it a competitive advantage in the infected gut.
A team led by investigators at Vanderbilt University Medical Center has discovered how C. diff (Clostridioides difficile) converts the poisonous compound 4-thiouracil, which could come from foods like broccoli, into a usable nutrient. Their findings, published March 25 in the journal Cell Host & Microbe, increase understanding of the molecular drivers of C. diff infection and point to novel therapeutic strategies.
C. diff causes about half a million infections in the U.S. each year, according to the Centers for Disease Control and Prevention. Factors that increase risk of C. diff infection include antibiotic use, age over 65, and recent stays in hospitals and other health care facilities.
Like other pathogens, C. diff must acquire nutrients to survive and grow.
We’re interested in trying to understand the nutrients that C. diff needs during infection, and how what you eat influences what C. diff eats in your gut.
Matthew Munneke, first author
Department of Pathology, Microbiology, and Immunology
Vanderbilt Institute for Infection, Immunology, and Inflammation
Vanderbilt University Medical Center, Nashville, TN.
The group focused on nucleotides — the building blocks of DNA and RNA — which are a class of nutrients that hasn’t been well studied for C. diff .
The researchers found that C. diff must acquire a certain type of nucleotides (pyrimidines) to cause infection, and they discovered an enzyme they named TudS (thiouracil desulfurase) that C. diff uses to salvage the pyrimidine nucleotide uracil from a related compound: 4-thiouracil.
They showed that 4-thiouracil gets incorporated into RNA and is toxic to resident gut microbes that do not have the TudS enzyme. In C. diff , however, TudS modifies and detoxifies 4-thiouracil, making it available as a nutrient. The researchers demonstrated that TudS contributes to C. diff “fitness” in mice fed 4-thiouracil and in a novel MiniBioreactor model that contains a community of bacteria isolated from human feces with added 4-thiouracil.
We think that 4-thiouracil metabolism is beneficial to C. diff because it acts as a nutrient to fuel the bacteria, and it also may inhibit neighboring bacteria, which would give C. diff a further competitive advantage within the gut environment.
Matthew Munneke
The TudS enzyme may represent a novel therapeutic target for treating C. diff infections. It is not present in many resident gut microbes (or in human cells), so an antimicrobial targeting it to kill C. diff might help preserve the healthy gut microbiota, he noted.
The researchers also showed that adding C. diff TudS to a probiotic strain of E. coli blunted C. diff ’s fitness advantage in an in vitro model.
It might be possible to use a probiotic with this enzyme to diminish C. diff ’s ability to thrive in the gut and push it out.
Matthew Munneke
F
Although the researchers showed that 4-thiouracil is present in the human gut, the source of this compound is unclear. Livestock that consume a diet rich in cruciferous vegetable family members (such as kale and other leafy greens, broccoli and cauliflower) have elevated levels of 4-thiouracil, and it is present in broccoli, both suggestive that a dietary source may contribute to the presence of 4-thiouracil in the human gut.
More research is needed to understand the source of 4-thiouracil, but if it comes from the diet, that could inform dietary interventions for C. diff infection.
Matthew Munneke
It’s not time to give up eating cruciferous vegetables though. In the healthy gut, some resident microbes contain a TudS-related enzyme and can likely convert 4-thiouracil into nutrients. These microbes may be missing in the C. diff -infected gut, Munneke said.
Other VUMC authors of the Cell Host & Microbe paper are Catherine Shelton, PhD, Darian Carroll, PhD, Nicole Kirchoff, PhD, Martin Douglass, PhD, M. Wade Calcutt, PhD, Katherine Gibson-Corley, DVM, PhD, Maribeth Nicholson, MD, MPH, and Mariana Byndloss, DVM, PhD. Collaborators at the University of Florida and Baylor College of Medicine contributed to the studies.
Highlights
Pyrimidine nucleotide synthesis is critical for Clostridioides difficile infection
A thiouracil desulfurase (TudS) enables C. difficile to utilize 4-thiouracil (4-TU)
TudS prevents 4-TU toxicity and incorporation into RNA by converting it to uracil
4-TU is present in the human gut and confers a fitness advantage to C. difficile
Summary
Nucleotides are essential building blocks for major cellular macromolecules and are critical for life. Consequently, bacterial pathogens must acquire or synthesize nucleotides during infection. Clostridioides difficile is the most common hospital-acquired gastrointestinal infection, and nutrient acquisition is critical for pathogenesis. However, the impact of nucleotide metabolism on C. difficile infection remains unclear. Here, we discover that 4-thiouracil (4-TU), a pyrimidine analog present in the human gut, is toxic to commensal bacteria. 4-TU hijacks the uracil salvage pathway for incorporation into RNA through the uracil phosphoribosyltransferase activity encoded by PyrR and Upp. C. difficile can salvage 4-TU as a pyrimidine source through the enzymatic action of a thiouracil desulfurase (TudS), thereby contributing to C. difficile fitness in mice fed 4-TU or MiniBioreactor models of infection containing exogenous 4-TU. Collectively, these results reveal a molecular mechanism for C. difficile to utilize a poisonous pyrimidine analog in the vertebrate gut to outcompete commensal microbes.
Graphical abstract
Munneke, Matthew J.; Yuan, Yifeng; Preisner, Eva C.; Shelton, Catherine D.; Carroll, Darian T.; Kirchoff, Nicole S.; Dickson, Ken P.; Cantu, Jose O.; Douglass, Martin V.; Calcutt, M. Wade; Gibson-Corley, Katherine N.; Nicholson, Maribeth R.; Byndloss, Mariana X.; Britton, Robert A.; de Crécy-Lagard, Valérie; Skaar, Eric P. A thiouracil desulfurase protects Clostridioides difficile RNA from 4-thiouracil incorporation, providing a competitive advantage in the gutCell Host & Microbe (2025) DOI:10.1016/j.chom.2025.03.001.
Of course, as anyone with even a basic grasp of evolutionary biology knows, all aspects of parasitic organisms and their ability to survive and thrive in hostile environments can be readily explained by the Theory of Evolution, which remains unrivalled in its ability to account for the observable evidence.
In contrast, intelligent-design creationists struggle to explain why an allegedly omniscient and omnibenevolent designer would deliberately create parasitic organisms. They must also explain why such a designer would equip these organisms with adaptations specifically to out-compete beneficial microbes in our microbiome, solely enabling parasites to replicate more effectively and cause greater suffering.
To date, I have yet to encounter any persuasive explanation for parasites or parasite-host arms races that could reasonably be attributed to the purposeful actions of a supremely intelligent and benevolent designer.
Not so long ago, it was commonly claimed that humans were exceptional due to their supposedly 'unique' ability to make and use tools. This assertion was often used to reinforce the idea that humans occupied a special position at the pinnacle of creation, justifying the biblical concept of human dominion over the rest of nature.
However, this claim was never credible to anyone observing nature carefully. It was largely promoted by religious authorities to foster a sense of human uniqueness and importance. This, in turn, reinforced belief in a creator god, supported the authority of religious institutions and their clerics, and justified their claims to the right to create laws governing human behaviour.
Scientific research has increasingly exposed the fallacy of this notion of human exceptionalism. Tool-making and tool use in humans are indeed more sophisticated than in other animals, but this ability is far from unique. Many other species demonstrate these abilities, notably chimpanzees—our closest living relatives. The widespread occurrence of tool use in nature strongly suggests this trait was present in a common ancestor we share with other primates. Furthermore, the independent evolution of tool use in species as diverse as birds, bees, and octopuses demonstrates that this capability is not unique to humans but rather a result of natural evolutionary processes.
Another human characteristic, traditionally cited by religious authorities as evidence for special creation and human exceptionalism, has, once again, been shown by science to be better explained as evidence of our evolutionary heritage within the natural world.
And today we have evidence that chimpanzees not only make and use tools but employ sophisticated 'engineering' skill in their choice of the right materials for their construction. It comes in the form of a paper published, open access, in iScience by a team of researchers led by Dr Alejandra Pascual-Garrido, of the School of Anthropology and Museum Ethnography, University of Oxford, UK.
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Analysing the cosmic microwave background in high definition has enabled researchers to confirm a simple model of the universe, ruling out many competing alternatives.
Creationists today face a distinct challenge compared to their predecessors from one or two centuries ago. They must continually devise ways to ignore or dismiss the relentless flow of scientific evidence disproving their beliefs, while simultaneously rationalizing the complete lack of evidence supporting claims of a young Earth, special creation through supernatural means, or the existence of a creator capable of producing a universe from nothing.
Individuals with a normal degree of intellectual honesty, when confronted with overwhelming evidence against their beliefs and a lack of supportive evidence, would naturally see this as grounds for doubt and reassessment. Creationists, however, appear undeterred, convinced that their personal beliefs override scientific evidence without the necessity for evidential justification.
Compounding their difficulties, scientists recently announced a significant advancement: they have mapped the cosmic microwave background radiation—the residual echo of the Big Bang—in unprecedented detail. Utilizing data from the Atacama Cosmology Telescope (ACT) collaboration, this new research reveals conditions in the universe as they existed only 380,000 years after the Big Bang, roughly 13.8 billion years ago.
On the scale of a human lifetime, this is the equivalent of a photograph of a now middle-aged person, taken one hour after they were born, and, in a confirmation of the principle of Occam's Razor, the simplest model is conformed as the correct model.
A significant issue with our immune system is that it is poorly "designed." If it were truly the product of an intelligent designer, as creationists claim, that designer would hardly be competent enough to design a simple household item, let alone a complex biological system.
Because our immune system is so disorganized and inefficient, multiple layers of complexity have evolved to mitigate its worst shortcomings. However, these added layers themselves remain prone to errors, as they reflect the same flawed foundation. The central problem arises because the immune system must balance two contradictory requirements: it needs to be sensitive enough to identify and eliminate genuine threats, yet not so sensitive that it mistakenly attacks the body's own tissues.
While an omnipotent, supremely intelligent designer should have easily resolved such a contradiction, the reality is that our immune system frequently fails on both counts. It often permits pathogens and parasites to invade, and it also frequently turns against the body itself, leading to autoimmune diseases such as lupus, diabetes, multiple sclerosis, arthritis, and kidney or liver failure, among numerous other debilitating conditions that cause immense suffering.
Like the whimsical contraptions created by cartoonist William Heath Robinson — complex machines built from objects originally intended for entirely different purposes - the mammalian immune system is not designed top-down from a clear blueprint. Rather, it's built up gradually from one makeshift adaptation piled onto another, each new solution attempting to compensate for the shortcomings of earlier ones. Eventually, this process results in a ramshackle system so intricate that its complexity itself creates new opportunities for failure. Such complexity is not indicative of intelligent, purposeful design, which would typically favour simplicity and efficiency. Instead, it reflects an ad hoc, utilitarian approach driven by evolutionary constraints and an inability to anticipate future challenges.
And of course, this embarrassment for creationism is made worse by the fact that, according to Michael J. Behe, pathogenic parasites such as E. coli and Plasmodium falciparum are examples of irreducible complexity, so are, in creationist circles, unarguable 'proof' of intelligent design, so the immune system is allegedly designed by the designer of these pathogens to protect us from them.
It seems creationists have no difficulty in believing the same designer would design parasites to make sick, then design a system to protect us from its pathogenic designs, and even though that system doesn't work very well, it is nevertheless evidence of supreme intelligence.
An artist’s depiction of an Asgard archaeon, based on cryo-electron tomography data: the cell body and appendages feature thread-like skeletal structures, similar to those found in complex cells with nuclei.
Graphic: Margot Riggi, Max Planck Institute of Biochemistry
Recent research indicates that the last universal common ancestor of complex (eukaryotic) cells, which encompass all multicellular plant and animal life, likely originated from the Asgard group of archaea. This ancestor is believed to have formed a symbiotic relationship with an alphaproteobacterium, which eventually evolved into the mitochondrion.
The initial nature of this symbiotic relationship—whether parasitic or predatory—remains uncertain. However, its establishment was pivotal in setting the evolutionary course that led to the diversity of life on Earth.
Compelling evidence supporting the Asgard archaea hypothesis has been uncovered by Professor Martin Pilhofer and his team at the Eidgenössische Technische Hochschule (ETH) Zurich, Switzerland. Their findings have been published in the journal Cell and is explained in an ETH news item by Peter Rüegg:
A point I never tire of making because it contrasts so vividly what the authors of the Bible thought the Universe was like and what science is showing us it is really like, is the ludicrously naive description of a small, flat planet with a dome over it, with the moon, sun and starts attached to the dome.
Don't take my word for it. Open your Bible and read the first few pages:
And God said, Let there be a firmament in the midst of the waters, and let it divide the waters from the waters. And God made the firmament, and divided the waters which were under the firmament from the waters which were above the firmament: and it was so. And God called the firmament Heaven. And the evening and the morning were the second day. And God said, Let the waters under the heaven be gathered together unto one place, and let the dry land appear: and it was so. And God called the dry land Earth; and the gathering together of the waters called he Seas: and God saw that it was good. (Genesis 1.6-10)
And God made two great lights; the greater light to rule the day, and the lesser light to rule the night: he made the stars also. And God set them in the firmament of the heaven to give light upon the earth, And to rule over the day and over the night, and to divide the light from the darkness: and God saw that it was good.(Genesis 1.16-18)
How the Bible's authors saw the Universe.
Now compare that with the view of just a small fragment of the universe that the European Space Agency's space telescope, Euclid, has revealed today:
Feathers provide a fascinating example of how evolution can repurpose structures over time. Initially evolving in response to one set of selective pressures, feathers later opened the door for entirely new functions unrelated to their original purpose.
Early feathers appeared among dinosaurs primarily as an adaptation for thermoregulation. Simple, filamentous feathers offered significantly better insulation than traditional reptilian scales, helping dinosaurs maintain stable body temperatures. Among bipedal theropod dinosaurs, these insulating feathers eventually evolved into more complex structures, freeing forelimbs to develop into wings. Feathers subsequently became specialized for powered flight, having first likely served intermediate functions such as display or gliding.
The presence of insulating feathers likely provided a survival advantage during the dramatic climate changes following the asteroid impact that marked the end of the Cretaceous period, approximately 66 million years ago, contributing to the extinction of non-avian dinosaurs.
This is why we see a clear progression through the fossil record, unlike what would be expected of an intelligent design process, where birds, complete with flight feathers, would be expected to make a sudden appearance without ancestry.
Today, feathers in modern birds retain their important role in thermoregulation while also facilitating flight and serving as display structures. Vibrant and diverse plumage has evolved under sexual selection pressures, playing a crucial role in mating rituals, mate choice, and reinforcing genetic isolation among closely related species, thereby preventing hybridization.
What information do you have on the fossil evidence for the evolution of feathers from those found on dinosaurs to those of flying birds?
Fossil evidence documenting the evolution of feathers from dinosaurs to modern birds has greatly improved in recent decades, particularly with discoveries from China. Key points include:
Filamentous Proto-feathers:
Early feather-like structures have been identified in fossils of non-avian dinosaurs such as Sinosauropteryx, which lived around 125 million years ago during the early Cretaceous period. These simple, filamentous structures, or "proto-feathers," were likely used primarily for insulation or display.
Complex Feather Structures:
More complex feathers resembling those of modern birds appear in later theropods. Fossils like Caudipteryx and Sinornithosaurus from the Late Jurassic and Early Cretaceous (around 160-120 million years ago) show branching structures with barbs and barbules, although they likely were incapable of sustained powered flight. These feathers probably served as insulation and were possibly used for display or gliding.
Flight and Transitional Forms:
Archaeopteryx, often cited as a key transitional fossil (approximately 150 million years old), possessed flight-capable feathers remarkably similar to those of modern birds, with asymmetrical vanes adapted for powered flight. However, Archaeopteryx still retained several dinosaurian traits, such as teeth, a long bony tail, and clawed fingers, clearly illustrating a transitional stage.
Microraptor and Gliding Flight:
Fossils of the theropod dinosaur Microraptor (approximately 120 million years ago) feature well-preserved feathers on all four limbs and the tail, forming a wing-like arrangement. This configuration strongly suggests it engaged in gliding flight, indicating experimentation in feathered flight prior to the development of the modern avian flight mechanism.
Preservation of Pigmentation:
Remarkably, some feathered dinosaur fossils, like Anchiornis, have preserved microscopic structures (melanosomes) indicating pigment patterns. These have provided insights into feather coloration and display functions, further highlighting evolutionary pressures beyond thermoregulation.
In short, the fossil record clearly demonstrates a gradual progression from simple insulating filaments to complex, asymmetrical flight feathers, directly connecting non-avian dinosaurs and modern birds.
But the key changes that lead to the evolution of feathers from reptilian scales had remained elusive until now. Now, two researchers from the University of Geneva (UNIGE), Switzerland, have uncovered a key role of a molecular signalling pathway (the Shh pathway) in their formation. They have published their findings in the journal, PLOS Biology, and explained them in a University of Geneve press release:
From dinosaurs to birds: the origins of feather formationUNIGE scientists reveal the key role of specific genes in feather development,
providing insights into their evolution from dinosaurs.
Feathers, essential for thermoregulation, flight, and communication in birds, originate from simple appendages known as proto-feathers, which were present in certain dinosaurs. By studying embryonic development of the chicken, two researchers from the University of Geneva (UNIGE) have uncovered a key role of a molecular signalling pathway (the Shh pathway) in their formation. This research, published in the journal PLOS Biology, provides new insights into the morphogenetic mechanisms that led to feather diversification throughout evolution.
Feathers are among the most complex cutaneous appendages in the animal kingdom. While their evolutionary origin has been widely debated, paleontological discoveries and developmental biology studies suggest that feathers evolved from simple structures known as proto-feathers. These primitive structures, composed of a single tubular filament, emerged around 200 million years ago in certain dinosaurs. Paleontologists continue to discuss the possibility of their even earlier presence in the common ancestor of dinosaurs and pterosaurs (the first flying vertebrates with membranous wings) around 240 million years ago.
The emergence of proto-feathers likely marked the first key step in feather evolution.
Proto-feathers are simple, cylindrical filaments. They differ from modern feathers by the absence of barbs and barbules, and by the lack of a follicle—an invagination at their base. The emergence of proto-feathers likely marked the first key step in feather evolution, initially providing thermal insulation and ornamentation before being progressively modified under natural selection to give rise to the more complex structures that enabled flight.
The laboratory of Michel Milinkovitch, professor at the Department of Genetics and Evolution in the Faculty of Science at UNIGE, studies the role of molecular signaling pathways (communication systems that transmit messages within and between cells), such as the Sonic Hedgehog (Shh) pathway, in the embryonic development of scales, hair, and feathers in modern vertebrates. In a previous study, the Swiss scientists stimulated the Shh pathway by injecting an activating molecule into the blood vessels of chicken embryos and observed the complete and permanent transformation of scales into feathers on the bird’s feet.
Recreating the first dinosaur proto-feathers
Since the Shh pathway plays a crucial role in feather development, we wanted to observe what happens when it is inhibited.
Dr. Rory L. Cooper, first-author.
Laboratory of Artificial and Natural Evolution (LANE)
Department of Genetics and Evolution
University of Geneva, Geneva, Switzerland.
By injecting a molecule that blocks the Shh signaling pathway on the 9th day of embryonic development – just before feather buds appear on the wings – the two researchers observed the formation of unbranched and non-invaginated buds, resembling the putative early stages of proto-feathers.
However, from the 14th day of embryonic development, feather morphogenesis partially recovered. Furthermore, although the chicks hatched with patches of naked skin, dormant subcutaneous follicles were autonomously reactivated, eventually producing chickens with normal plumage.
Our experiments show that while a transient disturbance in the development of foot scales can permanently turn them into feathers, it is much harder to permanently disrupt feather development itself. Clearly, over the course of evolution, the network of interacting genes has become extremely robust, ensuring the proper development of feathers even under substantial genetic or environmental perturbations. The big challenge now is to understand how genetic interactions evolve to allow for the emergence of morphological novelties such as proto-feathers.
Professor Michel C. Milinkovitch, corresponding author.
Laboratory of Artificial and Natural Evolution (LANE)
Department of Genetics and Evolution
University of Geneva, Geneva, Switzerland.
Abstract
The morphological intricacies of avian feathers make them an ideal model for investigating embryonic patterning and morphogenesis. In particular, the sonic hedgehog (Shh) pathway is an important mediator of feather outgrowth and branching. However, functional in vivo evidence regarding its role during feather development remains limited. Here, we demonstrate that an intravenous injection of sonidegib, a potent Shh pathway inhibitor, at embryonic day 9 (E9) temporarily produces striped domains (instead of spots) of Shh expression in the skin, arrests morphogenesis, and results in unbranched and non-invaginated feather buds—akin to proto-feathers—in embryos until E14. Although feather morphogenesis partially recovers, hatched treated chickens exhibit naked skin regions with perturbed follicles. Remarkably, these follicles are subsequently reactivated by seven weeks post-hatching. Our RNA-sequencing data and rescue experiment using Shh-agonism confirm that sonidegib specifically down-regulates Shh pathway activity. Overall, we provide functional evidence for the role of the Shh pathway in mediating feather morphogenesis and confirm its role in the evolutionary emergence and diversification of feathers.
Introduction
Avian feathers are intricate integumentary appendages, the forms of which vary substantially among species, across body areas, and between juvenile and adult stages. Understanding both the developmental and evolutionary mechanisms underpinning this morphological diversity has long fascinated biologists [1,2]. Although their evolutionary origin has been widely debated, feathers likely first appeared as simple cylindrical monofilaments in the common archosaurian ancestor of dinosaurs and pterosaurs during the Early Triassic [3–5]. Gradually, the morphology of these so-called ‘proto-feathers’ then increased in complexity to first produce simple plumulaceous down-type feathers (which lack a central shaft known as a rachis) and, later, to produce the more highly ordered pennaceous feathers [1,2], including bilaterally symmetric contour feathers and bilaterally asymmetric flight feathers (Fig 1A) [6]. This diversification was necessarily driven by modifications to the spatial expression of conserved developmental genes mediating feather morphogenesis, including the longitudinal domains of sonic hedgehog (Shh) expression observed during feather barb formation [7,8]. However, the precise effects of perturbing such molecular signaling throughout feather morphogenesis remain to be comprehensively investigated in vivo. More generally, feathers provide an ideal model for investigating molecular developmental patterning and morphogenesis [6,9].
.
Fig 1. Feather development in embryonic chicken.
(A) Hatched chickens at seven day post-hatching (7 dph) exhibit both pennaceous (with a central rachis) and plumulaceous (without a rachis) feather types. (B) At E9, LSFM reveals feather placodes covering most of the dorsum and expressing SHH protein (visualized by immunofluorescence). By E10, these placodes undergo considerable outgrowth and SHH becomes localized to the posterior tip of developing units. (C) Feather placodes first emerge on the posterior edge of the wings at E10, before propagating to cover the entire wing by E11. Optical sections reveal epidermal SHH immunofluorescence from E10 to E12. At E12, the longitudinal barbs associated with branching morphogenesis are visible, and by E13, invagination of the follicle is well-advanced. These units continue to develop until E14, at which point feather buds are elongated, keratinized, and exhibit a longitudinal pattern of cell density corresponding to the future feather barbs.
Although feathers arguably constitute the most complex and highly ordered group of integumentary appendages, their early embryonic development shares broad similarities in molecular signaling and morphogenesis with hairs and scales [10–13]. The morphological development of feathers begins with the emergence of an anatomical placode [12,14]—a local epidermal thickening and an underlying aggregation of dermal cells—associated with conserved patterns of gene expression [9]. Indeed, conserved placode signaling constitutes the foundation of integumentary appendages across diverse vertebrate clades, from the scales of sharks to the hairs of mammals [10–13]—with the exception of crocodile head scales [15,16]. The spatial distribution of placodes is widely considered to be mediated through paradigmatic chemical Turing reaction-diffusion dynamics, in which interactions between diffusing activatory and inhibitory morphogens give rise to stable periodic patterns [17–23]. Research regarding feather propagation in the chicken, and scale propagation in snakes, has revealed that chemical reaction-diffusion patterning is coupled with mechanical processes. Indeed, fibroblast growth factor (FGF) signaling triggers the aggregation of dermal cells which mechanically compress the overlying epidermis, thereby initiating subsequent local molecular signaling [23–26]. The outgrowth of feather placodes then gives rise to elongated and cylindrical feather buds, whilst invagination at their base gives rise to the follicle [6]. Finally, the feather bud divides into individual longitudinal filaments known as barbs, a process termed “branching morphogenesis”.
Previous research has also investigated the post-embryonic development of feathers. For example, the establishment of a Wnt3a signaling gradient appears to be required for the development of bilaterally symmetric contour feathers (but not radially symmetric plumulaceous feathers) during post-embryonic development [27]. Furthermore, coordinated adjustment of cell shape and adhesion, via the contraction of basal keratinocyte filopodia in the follicle, may contribute to the regulation of branching morphogenesis of pennaceous feathers [28]. Here, we investigate the embryonic development of the plumulaceous down-type feathers in the chicken.
The sonic hedgehog (Shh) pathway is a key regulator of diverse developmental processes [29,30]. Canonical Shh signaling begins with the SHH ligand binding to its receptor Patched (PTCH), thereby reversing the repression of the transmembrane protein Smoothened (SMO). The latter subsequently activates signaling via the transcription factor GLI, an intracellular zinc finger protein that triggers downstream transcription [31–33]. The Shh pathway plays multiple roles during feather development [34]. First, we have shown that transient in vivo agonism of Shh pathway signaling in chicken at embryonic day 11 (E11), through a single intravenous injection of smoothened agonist (SAG), triggers a complete and permanent transition from reticulate foot scales to feathers [35]. Remarkably, the resulting juvenile down-type ectopic feathers subsequently transition into adult regenerative bilaterally symmetric contour feathers, without the need for sustained SAG treatment, indicating that the over-expression of the Shh pathway at E11 induces a permanent shift in developmental fate of the corresponding placodes. Hence, the Shh pathway is clearly involved in the specification of avian skin appendages. Second, Shh mediates feather bud outgrowth by regulating interactions between the epidermis and mesenchyme [36] and may mediate the proliferation of dermal progenitor cells during morphogenesis [37]. Third, the relative level of Shh signaling within the posterior edge of the wing is involved in determining the location of specialized pennaceous flight feathers [38]. Fourth, feather branching morphogenesis in the embryonic chicken requires the spatial patterning of Shh through a reaction-diffusion mechanism that also involves the bone morphogenetic protein, Bmp2 [7,8]. In this proposed activator-inhibitor model, diffusing Shh activates its own transcription, as well as the transcription of Bmp2, which then inhibits Shh [8]. The stationary striped pattern of expression that arises from this mechanism provides a molecular template defining the position of individual feather barbs, i.e., Shh and Bmp2 are observed in characteristic longitudinal expression domains defining the edges of individual barb ridges [7,8]. Although replication-competent avian retrovirus infection has previously been used to manipulate the expression domains of Shh and Bmp2 that dictate feather barb formation [8], functional in vivo evidence regarding the roles of Shh during subsequent feather morphogenesis remains limited.
Here, we investigate feather development in the chicken. First, we present light sheet fluorescence microscopy (LSFM) imaging data regarding the normal patterning and morphogenesis of embryonic feathers. Next, we use precise intravenous in-ovo injections [35,39] of sonidegib to pharmacologically inhibit Shh pathway signaling during feather development at embryonic day 9 (E9), i.e., at the placodal stage preceding feather-bud outgrowth on the wings. This treatment temporarily modifies Shh expression to produce striped domains, temporarily arrests morphogenesis, and results in unbranched and non-invaginated feather buds—akin to putative proto-feathers—in embryos until E14. Although feather morphogenesis partially recovers, hatched sonidegib-treated chickens exhibit naked regions of the skin surface with dormant and perturbed follicles. Remarkably, these dormant follicles are subsequently reactivated before seven weeks of post-embryonic development. Using bulk RNA sequencing (RNAseq) and a rescue experiment (with SAG), we show that sonidegib specifically reduces Shh pathway signaling. Overall, we provide comprehensive functional evidence for the role of the Shh pathway in mediating feather morphogenesis in the chicken, supporting the hypothesis that modified Shh signaling has contributed to the evolutionary diversification of feathers.
Fig 2. WMISH reveals that sonidegib treatment arrests feather branching morphogenesis.
(A) The feathers of DMSO-treated control samples undergo normal patterning and morphogenesis [9], including the emergence of longitudinal Shh expression domains associated with branching morphogenesis from E12 to E13 [7,8]. (B) Samples treated with 100 µg sonidegib exhibit similar feather patterning to control samples but with slightly perturbed feather bud outgrowth (units are shorter across comparable embryonic stages). (C, D) Samples treated with either 200 or 300 µg sonidegib exhibit large striped expression domains of Shh at E10, demonstrating that these treatments temporarily modify the underlying reaction-diffusion dynamics. Normal spotted patterning recovers by E11 with Shh expression restricted to individual feather domains. However, subsequent morphogenesis from E11 to E13 remains perturbed as feather buds undergo substantially less outgrowth than in control samples. (D) At E13, samples treated with 300 µg sonidegib also reveal a reduction in longitudinal Shh expression domains associated with feather branching morphogenesis. (A–D, bottom row) As Shh WMISH becomes problematic after E13, only the morphological effect of sonidegib-induced dose-dependent reduction in feather bud outgrowth is shown at E14 (also shown in S1 Fig).
Fig 4. Naked skin regions in sonidegib-treated embryos contain perturbed follicles at E21.
(A) Control samples exhibit normal coverage of keratinized and filamentous feathers. LSFM of nuclear stained (TO-PRO-3) control samples reveals deeply embedded feather follicles from which highly keratinized feather shafts emerge, consisting of multiple individual barbs (white arrows). (B) The anterior wings of samples treated with 300 µg sonidegib exhibit naked regions of skin adorned with perturbed sub-epidermal feather follicles (blue arrows) that completely lack the outgrowth of feather buds (white arrows), and exhibit less tissue differentiation than observed in control samples. Therefore, sonidegib treatment does not abolish the patterning of feather follicles, but instead dramatically perturbs feather bud and follicle morphogenesis in these regions, resulting in naked areas of the skin.
Fig 6. SAG treatment rescues sonidegib-perturbed feather morphogenesis at E14.
Embryos were treated with SAG, a SMO agonist, together or following sonidegib delivery at E9. (A) Control embryos injected with DMSO at E9 exhibit normal elongated, filamentous feathers at E14. (B) Embryos treated at E9 with 300 µg sonidegib exhibit at E14 substantially reduced units that lack both branching and follicle invagination (blue arrow). (C) However, the combined injection of 300 µg sonidegib and 50 µg SAG at E9, prevents abnormal morphogenesis of feather buds. (D) Treatment with 100 µg SAG at E10 rescues feather morphogenesis in samples treated with 300 µg sonidegib at E9. (E) Treatment with 150 µg SAG at E11 only partially rescues feather morphogenesis in samples treated with 300 µg sonidegib at E9 (i.e., feather buds remain shorter at E14) probably because of a shorter recovery time (i.e., E11–E14). LSFM sections in the bottom panels reveal normal feather branching (white arrows) and follicle morphogenesis in the control and in all samples treated with both sonidegib and SAG, although samples treated with SAG at E11 exhibit reduced follicle invagination (E, bottom panel).
Fig 3. Sonidegib treatment arrests feather bud outgrowth and invagination.
We use LSFM of nuclear stained (TO-PRO-3) chicken wings at E14 to investigate the morphological effect of sonidegib treatment. (A) Control samples exhibit normal development of elongated feather buds with well-advanced follicles and visible barb ridges (white arrow). (B, C) We observe a dose-dependent effect of sonidegib treatment, with higher doses resulting in shorter feather buds and less advanced follicle development. (D) Samples treated with 300 µg sonidegib exhibit minimal feather bud outgrowth, the fusion of feather buds (white oval dotted outline), absent branching morphogenesis, and no follicle invagination (blue arrow).
Fig 5. Bulk RNA sequencing reveals that sonidegib treatment down-regulates Shh pathway signaling.
(A) Samples were injected with either DMSO (controls) or 300 µg sonidegib at E9. Wings were then dissected for RNA sequencing from E10 to E13, with four biological replicates used for each treatment at each stage. (B) From E11 onwards, PCA reveals a clear separation between control and treated samples. (C) Differentially expressed genes (DEGs) (filtered by a fold change of ≥1.4 and a false-discovery rate (FDR) adjusted P-value of ≤0.05) are shown in volcano plots for four embryonic stages. At E10, Shh pathway members Ptch1, Ptch2 and Gli1 are down-regulated. From E11 to E13, both Ptch2 and Shh are persistently down-regulated in treated samples relative to controls. See file S1 Data for the data underlying the graphs shown in the figure.
Fig 7. Post-embryonic effect of sonidegib treatment.
(A) Control samples exhibit complete coverage of (yellow) plumulaceous down-type feathers from 1 dph (top row) until 7 dph (second row). By 28 dph (third row), down-type feathers are observed transitioning into (white) pennaceous contour feathers. This process is complete by 49 dph (bottom row). (B) Samples treated with 100 µg sonidegib exhibit slightly more sparsely patterned feathers, with some patches of skin visible through their plumage. However, by 49 dph, their feather coverage appears indistinguishable from controls. (C, D) Samples treated with either 200 or 300 µg sonidegib exhibit large regions of naked skin on their backs, from 1 dph to 28 dph. Upon closer inspection, these naked regions exhibit regularly patterned skin elevations, which likely correspond to dormant subepidermal follicles (as shown in Fig 4B). By 49 dph, the feather coverage of samples treated with 200 or 300 µg sonidegib recovers and appears comparable to both controls and samples treated with 100 µg sonidegib.
Cooper RL, Milinkovitch MC (2025) In vivo sonic hedgehog pathway antagonism temporarily results in ancestral proto-feather-like structures in the chicken.PLoS Biol23(3): e3003061. https://doi.org/10.1371/journal.pbio.3003061
In summary, the authors of this paper demonstrated that inhibiting the Shh pathway caused the developing feathers of embryonic chicks to revert to a form resembling those of feathered dinosaurs. Such a result would be unexpected if feathers had been specifically and intelligently designed for birds, as creationists claim. Under an intelligent design scenario, blocking the Shh pathway should logically prevent feather formation entirely rather than reverting feathers to an ancestral state. Instead, this evidence strongly reinforces the evolutionary origin of modern bird feathers—represented here by the domestic chicken—as modified versions inherited from theropod dinosaur ancestors. Clearly, the evolutionary transition involved critical modifications to the Shh gene that controls this pathway in the developing embryo.
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