The Making of a Flight Feather: Bio-architectural Principles and Adaptation.

The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.

The mechano-chemical circuit drives skin organoid self-organization.

Stem cells in organoids self-organize into tissue patterns with unknown mechanisms. Here, we use skin organoids to analyze this process. Cell behavior videos show that the morphological transformation from multiple spheroidal units with morphogenesis competence (CMU) to planar skin is characterized by two abrupt cell motility-increasing events before calming down. The self-organizing processes are controlled by a morphogenetic module composed of molecular sensors, modulators, and executers. Increasing dermal stiffness provides the initial driving force (driver) which activates Yap1 (sensor) in epidermal cysts. Notch signaling (modulator 1) in epidermal cyst tunes the threshold of Yap1 activation. Activated Yap1 induces Wnts and MMPs (epidermal executers) in basal cells to facilitate cellular flows, allowing epidermal cells to protrude out from the CMU. Dermal cell-expressed Rock (dermal executer) generates a stiff force bridge between two CMU and accelerates tissue mixing via activating Laminin and ß1-integrin. Thus, this self-organizing coalescence process is controlled by a mechano-chemical circuit. Beyond skin, self-organization in organoids may use similar mechano-chemical circuit structures.

Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells.

Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: (i) continuous PKC inhibition and (ii) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin.

The chicken frizzle feather is due to an α-keratin (KRT75) mutation that causes a defective rachis.

Feathers have complex forms and are an excellent model to study the development and evolution of morphologies. Existing chicken feather mutants are especially useful for identifying genetic determinants of feather formation. This study focused on the gene F, underlying the frizzle feather trait that has a characteristic curled feather rachis and barbs in domestic chickens. Our developmental biology studies identified defects in feather medulla formation, and physical studies revealed that the frizzle feather curls in a stepwise manner. The frizzle gene is transmitted in an autosomal incomplete dominant mode. A whole-genome linkage scan of five pedigrees with 2678 SNPs revealed association of the frizzle locus with a keratin gene-enriched region within the linkage group E22C19W28_E50C23. Sequence analyses of the keratin gene cluster identified a 69 bp in-frame deletion in a conserved region of KRT75, an α-keratin gene. Retroviral-mediated expression of the mutated F cDNA in the wild-type rectrix qualitatively changed the bending of the rachis with some features of frizzle feathers including irregular kinks, severe bending near their distal ends, and substantially higher variations among samples in comparison to normal feathers. These results confirmed KRT75 as the F gene. This study demonstrates the potential of our approach for identifying genetic determinants of feather forms.

Autofluorescence microscopy as a non-invasive probe to characterize the complex mechanical properties of keratin-based integumentary organs: A feather paradigm.

Integumentary organs exhibit diverse morphologies and functions. The complex mechanical property of the architecture is mainly contributed by the ingenious multiscale assembly of keratins. A cross-scale characterization on keratin integration in an integument system will help us understand the principles on how keratin-based bio-architecture are built and function in nature. In this study, we used feather as a model integument organ. We develop autofluorescence (AF) microscopy to study the characteristics of its keratin assemblies over a wide range of length scales. The AF intensity of each feather component, following the hierarchy from the rachis to barb to barbule, decreased with the physical dimension. By combining the analysis of AF signal and tensile testing, we can probe regional material density and the associated mechanical strength in a composite feather. We further demonstrated that the AF micro-images could resolve subtle variations in the defective keratin assembly in feathers from frizzled chicken variants with a mutation in α-keratin 75. The distinction between AF patterns and the morphological features of feather components across different length scales indicated a synergetic interplay between material integration and complex morphogenesis during feather development. The work shows AF microscopy can serve as an easy and non-invasive approach to study multiscale keratin organizations and the associated bio-mechanical properties in diverse integumentary organs. This approach will facilitate our learning of many bio-inspired designs in diverse animal integumentary organs/appendages.

A quantitative image-based protocol for morphological characterization of cellular solids in feather shafts.

During morphogenesis, cellular sheets undergo dynamic folding to build functional forms. Here, we develop an image-based quantitative morphology field (QMorF) protocol that quantifies the morphological features of cellular structures and associated distributions. Using feather shafts with different rigidities as examples, QMorF performs coarse-graining statistical measurements of the fitted cellular objects over a micro-image stack, revealing underlying mechanical coupling and developmental clues. These images give intuitive representations of mechanical forces and should be useful for analyzing tissue images showing clear cellular features. For complete details on the use and execution of this protocol, please refer to Chang et al. (2019).

Making region-specific integumentary organs in birds: evolution and modifications.

Birds are the most diversified terrestrial vertebrates due to highly diverse integumentary organs that enable robust adaptability to various eco-spaces. Here we show that this complexity is built upon multi-level regional specifications. Across-the-body (macro-) specification includes the evolution of beaks and feathers as new integumentary organs that are formed with regional specificity. Within-an-organ (micro-) specification involves further modifications of organ shapes. We review recent progress in elucidating the molecular mechanisms underlying feather diversification as an example. (1) ß-Keratin gene clusters are regulated by typical enhancers or high order chromatin looping to achieve macro- and micro-level regional specification, respectively. (2) Multi-level symmetry-breaking of feather branches confers new functional forms. (3) Complex color patterns are produced by combinations of macro-patterning and micro-patterning processes. The integration of these findings provides new insights toward the principle of making a robustly adaptive bio-interface.

Variations of Mesozoic feathers: Insights from the morphogenesis of extant feather rachises.

The rachises of extant feathers, composed of dense cortex and spongy internal medulla, are flexible and light, yet stiff enough to withstand the load required for flight, among other functions. Incomplete knowledge of early feathers prevents a full understanding of how cylindrical rachises have evolved. Bizarre feathers with unusually wide and flattened rachises, known as "rachis-dominated feathers" (RDFs), have been observed in fossil nonavian and avian theropods. Newly discovered RDFs embedded in early Late Cretaceous Burmese ambers (about 99 million year ago) suggest the unusually wide and flattened rachises mainly consist of a dorsal cortex, lacking a medulla and a ventral cortex. Coupled with findings on extant feather morphogenesis, known fossil RDFs were categorized into three morphotypes based on their rachidial configurations. For each morphotype, potential developmental scenarios were depicted by referring to the rachidial development in chickens, and relative stiffness of each morphotype was estimated through functional simulations. The results suggest rachises of RDFs are developmentally equivalent to a variety of immature stages of cylindrical rachises. Similar rachidial morphotypes documented in extant penguins suggest that the RDFs are not unique to Mesozoic theropods, although they are likely to have evolved independently in extant penguins.

Fabrication of monolayer of polymer/nanospheres hybrid at a water-air interface.

A new type of polymer-assisted self-assembly of nanospheres at a water-air interface was uncovered. By adding merely 1-3 ppm of polyethylene oxide in the water, the polystyrene nanospheres, applicable to diameters ranging from 100 nm to 1 µm, were found to gradually move closer to each other and eventually form a close-packed structure confirmed from its diffraction pattern. As it turns out, polyethylene oxides are adsorbed onto the surface of polystyrene nanospheres, giving rise to the effective screening of coulomb repulsive force between nanospheres followed by the onset of polymer-bridging effect as demonstrated from the strong suppression of Brownian motion. The resulting monolayer of close-packed polymer/nanospheres hybrid at the water-air interface with area size more than 1 cm(2) are robust and can be transferred to a substrate of any kind without serious breaking due to surface tension tearing. Our finding may provide a further extension to the scope of nanosphere lithography technique.