Prof Michel C Milinkovitch

Vertebrate skin appendages are diverse micro-organs such as scales, feathers, and hair. These units typically develop from placodes, whose spatial patterning involves conserved chemical reaction-diffusion dynamics. Crocodile head scales are a spectacular exception to this paradigm, as they instead arise from a mechanically dominated process of compressive folding driven by constrained skin growth. Here, we reveal that chemical versus mechanical processes pattern tortoise scales in different regions of their head. Indeed, we show that placode-derived scales emerge across the peripheral head surfaces while remaining absent from the central dorsal region where scales subsequently form through a mechanical folding process. Using light sheet microscopy, we build a three-dimensional mechanical model that qualitatively recapitulates the diversity of scale patterns observed in this central head region in different tortoise species. Overall, our analyses indicate that mechanical head-scale patterning likely arose before the divergence between Testudinata and Archosauria, and was subsequently lost in birds.

Many examples of self-organized embryonic patterning can be attributed to chemically mediated systems comprising interacting morphogens. However, mechanical patterning also contributes to the emergence of biological forms. For example, various studies have demonstrated that diverse patterns arise from elastic instabilities associated with the constrained growth of soft materials, which generate wrinkles, creases and folds. Here, we show that between days 12 and 13 of development, transient experimentally increased activity of the sonic hedgehog pathway in the chicken embryo, through a single intravenous injection of smoothened agonist (SAG), abolishes the Turing-like chemical patterning of reticulate scales on the ventral footpad and promotes a transition to mechanical labyrinthine skin folding. Using hybridization, nanoindentation and labelling of proliferating cells, we confirm that skin surface folding is associated with the loss of signalling placode pre-patterning as well as increased epidermal growth and stiffness. Using additional hydrocortisone treatments, we also demonstrate that experimentally induced hyper-keratinization of the skin mechanically restricts SAG-induced folding. Finally, we verify our experimental findings with mechanical growth simulations built from volumetric light sheet fluorescence microscopy data. Overall, we reveal that pharmacological perturbation of the underlying gene regulatory network can abolish chemical skin appendage patterning and replace it with self-organized mechanical folding.

Snakes exhibit a broad variety of adaptive colors and color patterns, generated by the spatial arrangement of chromatophores, but little is known of the mechanisms responsible for these spectacular traits. Here, we investigate a mono-locus trait with two recessive alleles, motley and stripe, that both cause pattern aberrations in the corn snake.

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.

Amniote integumentary appendages constitute a diverse group of micro-organs, including feathers, hair and scales. These structures typically develop as genetically controlled units1, the spatial patterning of which emerges from a self-organized chemical Turing system2,3 with integrated mechanical feedback4,5. The seemingly purely mechanical patterning of polygonal crocodile head scales provides an exception to this paradigm6. However, the nature and origin of the mechanical stress field driving this patterning remain unclear. Here, using precise in ovo intravenous injections of epidermal growth factor protein, we generate Nile crocodile embryos with substantially convoluted head skin, as well as hatchlings with smaller polygonal head scales resembling those of caimans. We then use light-sheet fluorescence microscopy to quantify embryonic tissue-layer geometry, collagen architecture and the spatial distribution of proliferating cells. Using these data, we build a phenomenological three-dimensional mechanical growth model that recapitulates both normal and experimentally modified patterning of crocodile head scales. Our experiments and numerical simulations demonstrate that crocodile head scales self-organize through compressive folding, originating from near-homogeneous skin growth with differential stiffness of the dermis versus the epidermis. Our experiments and theoretical morphospace analyses indicate that variation in embryonic growth and material properties of skin layers provides a simple evolutionary mechanism that produces a diversity of head-scale patterns among crocodilian species.

Amniote integumentary appendages constitute a diverse group of micro-organs, including feathers, hair and scales. These structures typically develop as genetically controlled units, the spatial patterning of which emerges from a self-organized chemical Turing system with integrated mechanical feedback. The seemingly purely mechanical patterning of polygonal crocodile head scales provides an exception to this paradigm. However, the nature and origin of the mechanical stress field driving this patterning remain unclear. Here, using precise in ovo intravenous injections of epidermal growth factor protein, we generate Nile crocodile embryos with substantially convoluted head skin, as well as hatchlings with smaller polygonal head scales resembling those of caimans. We then use light-sheet fluorescence microscopy to quantify embryonic tissue-layer geometry, collagen architecture and the spatial distribution of proliferating cells. Using these data, we build a phenomenological three-dimensional mechanical growth model that recapitulates both normal and experimentally modified patterning of crocodile head scales. Our experiments and numerical simulations demonstrate that crocodile head scales self-organize through compressive folding, originating from near-homogeneous skin growth with differential stiffness of the dermis versus the epidermis. Our experiments and theoretical morphospace analyses indicate that variation in embryonic growth and material properties of skin layers provides a simple evolutionary mechanism that produces a diversity of head-scale patterns among crocodilian species.

The glabrous skin of the rhinarium (naked nose) of many mammalian species exhibits a polygonal pattern of grooves that retain physiological fluid, thereby keeping their nose wet and, among other effects, facilitating the collection of chemosensory molecules. Here, we perform volumetric imaging of whole-mount rhinaria from sequences of embryonic and juvenile cows, dogs, and ferrets. We demonstrate that rhinarial polygonal domains are not placode-derived skin appendages but arise through a self-organized mechanical process consisting of the constrained growth and buckling of the epidermal basal layer, followed by the formation of sharp epidermal creases exactly facing an underlying network of stiff blood vessels. Our numerical simulations show that the mechanical stress generated by excessive epidermal growth concentrates at the positions of vessels that form rigid base points, causing the epidermal layers to move outward and shape domes-akin to arches rising against stiff pillars. Remarkably, this gives rise to a larger length scale (the distance between the vessels) in the surface folding pattern than would otherwise occur in the absence of vessels. These results hint at a concept of "mechanical positional information" by which material properties of anatomical elements can impose local constraints on an otherwise globally self-organized mechanical pattern. In addition, our analyses of the rhinarial patterns in cow clones highlight a substantial level of stochasticity in the pre-pattern of vessels, while our numerical simulations also recapitulate the disruption of the folding pattern in cows affected by a hereditary disorder that causes hyperextensibility of the skin.

In 1952, Alan Turing published the reaction-diffusion (RD) mathematical framework, laying the foundations of morphogenesis as a self-organized process emerging from physicochemical first principles. Regrettably, this approach has been widely doubted in the field of developmental biology. First, we summarize Turing's line of thoughts to alleviate the misconception that RD is an artificial mathematical construct. Second, we discuss why phenomenological RD models are particularly effective for understanding skin color patterning at the meso/macroscopic scales, without the need to parameterize the profusion of variables at lower scales. More specifically, we discuss how RD models () recapitulate the diversity of actual skin patterns, () capture the underlying dynamics of cellular interactions, () interact with tissue size and shape, () can lead to ordered sequential patterning, () generate cellular automaton dynamics in lizards and snakes, () predict actual patterns beyond their statistical features, and () are robust to model variations. Third, we discuss the utility of linear stability analysis and perform numerical simulations to demonstrate how deterministic RD emerges from the underlying chaotic microscopic agents.

Two influential concepts in tissue patterning are Wolpert's positional information and Turing's self-organized reaction-diffusion (RD). The latter establishes the patterning of hair and feathers. Here, our morphological, genetic, and functional-by CRISPR-Cas9-mediated gene disruption-characterization of wild-type versus "scaleless" snakes reveals that the near-perfect hexagonal pattern of snake scales is established through interactions between RD in the skin and somitic positional information. First, we show that ventral scale development is guided by hypaxial somites and, second, that ventral scales and epaxial somites guide the sequential RD patterning of the dorsolateral scales. The RD intrinsic length scale evolved to match somite periodicity, ensuring the alignment of ribs and scales, both of which play a critical role in snake locomotion.

We present a technique for precise drug delivery into the vascular system of developing amniote embryos via injection into chorioallantoic veins underlying the eggshell membrane. We describe steps for incubating and candling eggs, removing the shell to expose underlying veins, and precise intravenous injection. In addition to chicken embryos, this protocol is applicable to other amniote species that lay hard-shell eggs, including crocodiles and tortoises. This technique is rapid, is reproducible, is of low cost, and will provide an important resource for developmental biologists. For complete details on the use and execution of this protocol, please refer to Cooper & Milinkovitch..