Neil H. Shubin

The fin-to-limb transition in vertebrate evolution has been central to the study of how development underlies evolutionary change. In this context, the functional analysis of Hox gene regulation to infer evolutionary trajectories has been critical to explain the origin of new features. In tetrapods, the transcription of Hoxd genes in developing digits depends on a set of enhancers forming a large regulatory landscape^1,2. The presence of a syntenic counterpart in zebrafish, which lacks digits, suggests deep homology³ or shared developmental foundations underlying distal fin and limbs. However, how this regulatory program evolved has remained unresolved. We genetically evaluated the function of the zebrafish Hoxd regulatory landscapes by comparatively assessing the effects of their full deletions. We show that, unlike in mice, deletion of these regions in fish does not disrupt hoxd gene transcription during distal fin development. By contrast, we found that this deficiency leads to the loss of expression within the cloaca, a structure related by ancestry to the mammalian urogenital sinus, and that distal hox13 genes are essential for correct cloacal formation. Because Hoxd gene regulation in the mouse urogenital sinus relies on enhancers located in this same chromatin domain that controls digit development, we propose that the current regulatory landscape active in distal limbs was co-opted as a whole in tetrapods from a pre-existing cloacal regulatory machinery.

Background: To understand cellular morphology, biologists have relied on traditional optical microscopy of tissues combined with tissue clearing protocols to image structures deep within tissues. Unfortunately, these protocols often struggle to retain cell boundary markers, especially at high enough resolutions necessary for precise cell segmentation. This limitation affects the ability to study changes in cell shape during major developmental events. Results: We introduce a method that preserves cell boundary markers and matches the refractive index of tissues with water. This technique enables the use of high‐magnification, long working distance water‐dipping objectives that provide sub‐micron resolution images. We subsequently segment individual cells using a trained neural network segmentation model. These segmented images facilitate quantification of cell properties of the entire three‐dimensional tissue. As a demonstration, we examine mandibles of transgenic mice that express fluorescent proteins in their cell membranes and extend this technique to a non‐model animal, the catshark, investigating its dental lamina and dermal denticles—invaginating and evaginating ectodermal structures, respectively. This technique provides insight into the mechanical environment that cells experience during developmental transitions. Conclusions: This pipeline, named MORPHOVIEW, provides a powerful tool to quantify in high throughput the 3D structures of cells and tissues during organ morphogenesis.

The earliest record of tooth antecedents and the tissue dentine^1,2, an early-vertebrate novelty, has been controversially represented by fragmentary Cambrian fossils identified as Anatolepis heintzi^3,4,5. Anatolepis exoskeletons have the characteristic tubules of dentine that prompted their interpretation as the first precursors of teeth³, known as odontodes. Debates over whether Anatolepis is a legitimate vertebrate^6,7,8 have arisen because of limitations in imaging and the lack of comparative exoskeletal tissues. Here, to resolve this controversy and understand the origin of dental tissues, we synchrotron-scanned diverse extinct and extant vertebrate and invertebrate exoskeletons. We find that the tubules of Anatolepis have been misidentified as dentine tubules and instead represent aglaspidid arthropod sensory sensilla structures^9,10. Synchrotron scanning reveals that deep ultrastructural similarities between odontodes and sensory structures also extend to definitive vertebrate tissues. External odontodes of the Ordovician vertebrate Eriptychius^11,12,13 feature large dentine tubules¹ that are morphologically convergent with invertebrate sensilla. Immunofluorescence analysis shows that the external odontodes of extant chondrichthyans and teleosts retain extensive innervation suggestive of a sensory function akin to teeth^14,15,16. These patterns of convergence and innervation reveal that dentine evolved as a sensory tissue in the exoskeleton of early vertebrates, a function retained in modern vertebrate teeth¹⁶. Middle-Ordovician fossils now represent the oldest known evidence for vertebrate dental tissues.

Synovial joints, characterized by reciprocally congruent and lubricated articular surfaces separated by a cavity, can simultaneously provide mobility and load bearing. Here, we study the early evolution of synovial joints by examining the morphological, genetic, and molecular features required for the development and function of the joints in elasmobranchs and cyclostomes. We show the presence of cavitated and articulated joints in the skeleton of elasmobranchs, such as the little skate (Leucoraja erinacea) and bamboo shark (Chiloscyllium plagiosum). However, our results do not support the presence of articular cavities between cartilaginous elements in cyclostomes such as sea lampreys (Petromyozon marinus) and hagfish (Myxine glutinosa). Immunostaining reveals the expression of lubrication-related proteoglycans like aggrecan and glycoproteins such as hyaluronic acid receptor (CD44) at the articular surfaces in little skates. Analysis of joint development in little skate embryos shows the expression of growth differentiation factor-5 (Gdf5) and β-catenin at the joint interzones like tetrapods. Muscle paralysis in little skate embryos leads to joint fusion, suggesting that muscle activity is necessary for the formation of synovial cavity and development of normal articular surfaces, in a manner similar to zebrafish and tetrapods. Together, these data suggest that synovial joints originated in the common ancestor of extant gnathostomes. A review of fossils from the extinct clades along the gnathostome stem suggests that joints with reciprocally articulating surfaces arose in the dermal skeleton of the common ancestor of all jawed vertebrates. Synovial joints in cartilaginous tissue were a subsequent gnathostome innovation.

Paleontologists must confront the challenge of studying the forms and functions of extinct species for which data from preserved fossils are extremely limited, yielding only a fragmented picture of life in deep time. In response to this hurdle, we describe the nascent field of paleoinspired robotics, an innovative method that builds upon established techniques in bioinspired robotics, enabling the exploration of the biology of ancient organisms and their evolutionary trajectories. This Review presents ways in which robotic platforms can fill gaps in existing research using the exemplars of notable transitions in vertebrate locomotion. We examine recent case studies in experimental paleontology, highlighting substantial contributions made by engineering and robotics techniques, and further assess how the efficient application of robotic technologies in close collaboration with paleontologists and biologists can offer additional insights into the study of evolution that were previously unattainable.

In recent years, we have seen a significant increase in our understanding of the mechanisms of development, regeneration, and healing of the respiratory system. However, most of these studies have been limited by their focus on mammalian systems. Here, we aimed to identify the underlying molecular mechanisms that are active during lung growth and tissue repair in amphibians, specifically Xenopus tropicalis ( X. tropicalis ). First, we analyzed the stem cell composition and signaling pathways that are active in epithelial and mesenchymal cells during lung growth. Then, we established a protocol for lung injury to assess the types of stem cells underlying tissue repair. In mammals, Sftpc+ (AT2) cells are alveolar stem cells that can differentiate to Krt8+ cells during lung homeostasis and post-injury repair. In this study, we identified Sftpc+ cells and Krt8+ cells, along with the activity of key developmental signaling pathways, Hippo and Wnt, during lung maturation at post-metamorphosis stages. We then established a protocol for lung injury using chemically induced injury with bleomycin, which damages the lung through oxidative stress. The results show an elevation in collagen post-injury, indicating bleomycin’s effect in causing lung fibrosis. X. tropicalis froglets survived 42 days post-injury, with a continuous decrease in fibrosis. To explore this effect, we analyzed the distribution of lung stem cells; Sox9 protein levels and Sftp gene expression were downregulated at the alveoli 42 days post-injury. The decrease in stem cell marker expression 42 days post-injury suggests they are differentiating as part of the healing process. Nevertheless, we could still detect them after a few weeks of healing. These results suggest that X. tropicalis has a regenerative capacity for lung tissue repair and that the same signaling pathways and stem cells are active in both amphibians and mammalian lungs during lung growth, regeneration, and healing. These findings show for the first time the physiological similarities between the anuran and the mammalian lung during growth and tissue repair processes, suggesting X. tropicalis as a potential animal model to study lung regeneration.

Background: Salamanders are the only tetrapods that exhibit the ability to fully regenerate limbs. The axolotl, a neotenic salamander, has become the model organism for regeneration research. Great advances have been made providing a detailed understanding of the morphological and molecular processes involved in limb regeneration. However, it remains largely unknown how limb regeneration varies across salamanders and how factors like variable life histories, ecologies, and limb functions have influenced and shaped regenerative capacities throughout evolution.Results: This study focuses on six species of plethodontid salamanders representing distinct life histories and habitats. Specimens were examined for regeneration ability after bite injuries as well as after controlled amputations. Morphological investigations revealed great regenerative abilities in all investigated species and frequent anatomical limb anomalies. Correlations were observed with respect to speed of regeneration and habitat.Conclusions: Investigating regeneration in non‐model salamander taxa is essential for disentangling shared features of the regeneration process versus those that may be more taxon‐specific. Gaining insights into variable aspects of regeneration under natural conditions and after conspecific biting rather than controlled amputations adds important new datapoints for understanding the evolutionary framework of regeneration and provides a broader context for interpreting findings made in the model organism axolotl.

Evolutionary novelties entail the origin of morphologies that enable new functions. These features can arise through changes to gene function and regulation. One key novelty is the fused rod at the end of the vertebral column in anurans, the urostyle. This feature is composed of a coccyx and a hypochord, both of which ossify during metamorphosis. To elucidate the genetic basis of these features, we used laser capture microdissection of these tissues and did RNA-seq and ATAC-seq at three developmental stages in tadpoles of Xenopus tropicalis . RNA-seq reveals that the coccyx and hypochord have two different molecular signatures. Neuronal ( TUBB3 ) and muscle markers ( MYH3 ) are upregulated in coccygeal tissues, whereas T-box genes ( TBXT , TBXT.2 ), corticosteroid stress hormones ( CRCH.1 ) and matrix metallopeptidases ( MMP1 , MMP8 and MMP13 ) are upregulated in the hypochord. ATAC-seq reveals potential regulatory regions that are observed in proximity to candidate genes that regulate ossification identified from RNA-seq. Even though an ossifying hypochord is only present in anurans, this ossification between the vertebral column and the notochord resembles a congenital vertebral anomaly seen prenatally in humans caused by an ectopic expression of the TBXT / TBXT.2 gene. This work opens the way to functional studies that can elucidate anuran bauplan evolution.

For decades, biologists have relied on confocal microscopy to understand cellular morphology and fine details of tissue structure. However, traditional confocal microscopy of tissues faces limited light penetration, typically less than 100 µm, due to tissue opacity. To address this challenge, researchers have developed tissue clearing protocols compatible with confocal microscopy. Unfortunately, these protocols often struggle to retain cell boundary markers, especially at high resolutions necessary for precise cell segmentation. In this work, we introduce a method that preserves cell boundary markers and matches the refractive index of tissues with water. This technique enables the use of high-magnification, long working distance water-dipping objectives. The sub-micron resolutions achieved with this approach allows us to automatically segment each individual cell using a trained neural network segmentation model. These segmented images facilitate the quantification of cell properties and morphology of the entire three-dimensional tissue. As a demonstration of this methodology, we first examine mandibles of transgenic mice that express fluorescent proteins in their cell membranes. We then extend this technique to a non-model animal, the catshark, investigating the cellular properties of its dental lamina and dermal denticles – invaginating and evaginating ectodermal structures, respectively. Our technique thus provides a powerful tool to quantify in high throughput the 3D structures of cells and tissues during organ morphogenesis.