scRNA-sequencing in chick suggests a probabilistic model for cell fate allocation at the neural plate border.

The vertebrate 'neural plate border' is a transient territory located at the edge of the neural plate containing precursors for all ectodermal derivatives: the neural plate, neural crest, placodes and epidermis. Elegant functional experiments in a range of vertebrate models have provided an in-depth understanding of gene regulatory interactions within the ectoderm. However, these experiments conducted at tissue level raise seemingly contradictory models for fate allocation of individual cells. Here, we carry out single cell RNA sequencing of chick ectoderm from primitive streak to neurulation stage, to explore cell state diversity and heterogeneity. We characterise the dynamics of gene modules, allowing us to model the order of molecular events which take place as ectodermal fates segregate. Furthermore, we find that genes previously classified as neural plate border 'specifiers' typically exhibit dynamic expression patterns and are enriched in either neural, neural crest or placodal fates, revealing that the neural plate border should be seen as a heterogeneous ectodermal territory and not a discrete transitional transcriptional state. Analysis of neural, neural crest and placodal markers reveals that individual NPB cells co-express competing transcriptional programmes suggesting that their ultimate identify is not yet fixed. This population of 'border located undecided progenitors' (BLUPs) gradually diminishes as cell fate decisions take place. Considering our findings, we propose a probabilistic model for cell fate choice at the neural plate border. Our data suggest that the probability of a progenitor's daughters to contribute to a given ectodermal derivative is related to the balance of competing transcriptional programmes, which in turn are regulated by the spatiotemporal position of a progenitor.

A gene regulatory network for neural induction.

During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen's node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.

An epithelial signalling centre in sharks supports homology of tooth morphogenesis in vertebrates.

Development of tooth shape is regulated by the enamel knot signalling centre, at least in mammals. Fgf signalling regulates differential proliferation between the enamel knot and adjacent dental epithelia during tooth development, leading to formation of the dental cusp. The presence of an enamel knot in non-mammalian vertebrates is debated given differences in signalling. Here, we show the conservation and restriction of fgf3, fgf10, and shh to the sites of future dental cusps in the shark (Scyliorhinus canicula), whilst also highlighting striking differences between the shark and mouse. We reveal shifts in tooth size, shape, and cusp number following small molecule perturbations of canonical Wnt signalling. Resulting tooth phenotypes mirror observed effects in mammals, where canonical Wnt has been implicated as an upstream regulator of enamel knot signalling. In silico modelling of shark dental morphogenesis demonstrates how subtle changes in activatory and inhibitory signals can alter tooth shape, resembling developmental phenotypes and cusp shapes observed following experimental Wnt perturbation. Our results support the functional conservation of an enamel knot-like signalling centre throughout vertebrates and suggest that varied tooth types from sharks to mammals follow a similar developmental bauplan. Lineage-specific differences in signalling are not sufficient in refuting homology of this signalling centre, which is likely older than teeth themselves.

Development and regeneration of the crushing dentition in skates (Rajidae).

Sharks and rays (elasmobranchs) have the remarkable capacity to continuously regenerate their teeth. The polyphyodont system is considered the ancestral condition of the gnathostome dentition. Despite this shared regenerative ability, sharks and rays exhibit dramatic interspecific variation in their tooth morphology. Ray (batoidea) teeth typically constitute crushing pads of flattened teeth, whereas shark teeth are pointed, multi-cuspid units. Although recent research has addressed the molecular development of the shark dentition, little is known about that of the ray. Furthermore, how dental diversity within the elasmobranch lineage is achieved remains unknown. Here, we examine dental development and regeneration in two Batoid species: the thornback skate (Raja clavata) and the little skate (Leucoraja erinacea). Using in situ hybridization and immunohistochemistry, we examine the expression of a core gnathostome dental gene set during early development of the skate dentition and compare it to development in the shark. Elasmobranch tooth development is highly conserved, with sox2 likely playing an important role in the initiation and regeneration of teeth. Alterations to conserved genes expressed in an enamel knot-like signalling centre may explain the morphological diversity of elasmobranch teeth, thereby enabling sharks and rays to occupy diverse dietary and ecological niches.

The Dental Lamina: An Essential Structure for Perpetual Tooth Regeneration in Sharks.

In recent years, nonclassical models have emerged as mainstays for studies of evolutionary, developmental, and regenerative biology. Genomic advances have promoted the use of alternative taxa for the study of developmental biology, and the shark is one such emerging model vertebrate. Our research utilizes the embryonic shark (Scyliorhinus canicula) to characterize key developmental and regenerative processes that have been overlooked or not possible to study with more classic developmental models. Tooth development is a major event in the construction of the vertebrate body plan, linked in part with the emergence of jaws. Early development of the teeth and morphogenesis is well known from the murine model, but the process of tooth redevelopment and regeneration is less well known. Here we explore the role of the dental lamina in the development of a highly regenerative dentition in sharks. The shark represents a polyphyodont vertebrate with continuously repeated whole tooth regeneration. This is presented as a major developmental shift from the more derived renewal process that the murine model offers, where incisors exhibit continuous renewal and growth of the same tooth. Not only does the shark offer a study system for whole unit dental regeneration, it also represents an important model for understanding the evolutionary context of vertebrate tooth regeneration.

Evolution and Developmental Diversity of Skin Spines in Pufferfishes.

Teleost fishes develop remarkable varieties of skin ornaments. The developmental basis of these structures is poorly understood. The order Tetraodontiformes includes diverse fishes such as the ocean sunfishes, triggerfishes, and pufferfishes, which exhibit a vast assortment of scale derivatives. Pufferfishes possess some of the most extreme scale derivatives, dermal spines, erected during their characteristic puffing behavior. We demonstrate that pufferfish scale-less spines develop through conserved gene interactions that underlie general vertebrate skin appendage formation, including feathers and hair. Spine development retains conservation of the EDA (ectodysplasin) signaling pathway, important for the development of diverse vertebrate skin appendages, including these modified scale-less spines of pufferfish. Further modification of genetic signaling from both CRISPR-Cas9 and small molecule inhibition leads to loss or reduction of spine coverage, providing a mechanism for skin appendage diversification observed throughout the pufferfishes. Pufferfish spines have evolved broad variations in body coverage, enabling adaptation to diverse ecological niches.

An ancient Turing-like patterning mechanism regulates skin denticle development in sharks.

Vertebrates have a vast array of epithelial appendages, including scales, feathers, and hair. The developmental patterning of these diverse structures can be theoretically explained by Alan Turing's reaction-diffusion system. However, the role of this system in epithelial appendage patterning of early diverging lineages (compared to tetrapods), such as the cartilaginous fishes, is poorly understood. We investigate patterning of the unique tooth-like skin denticles of sharks, which closely relates to their hydrodynamic and protective functions. We demonstrate through simulation models that a Turing-like mechanism can explain shark denticle patterning and verify this system using gene expression analysis and gene pathway inhibition experiments. This mechanism bears remarkable similarity to avian feather patterning, suggesting deep homology of the system. We propose that a diverse range of vertebrate appendages, from shark denticles to avian feathers and mammalian hair, use this ancient and conserved system, with slight genetic modulation accounting for broad variations in patterning.

Spatially restricted dental regeneration drives pufferfish beak development.

Vertebrate dentitions are extraordinarily diverse in both morphology and regenerative capacity. The teleost order Tetraodontiformes exhibits an exceptional array of novel dental morphologies, epitomized by constrained beak-like dentitions in several families, i.e., porcupinefishes, three-toothed pufferfishes, ocean sunfishes, and pufferfishes. Modification of tooth replacement within these groups leads to the progressive accumulation of tooth generations, underlying the structure of their beaks. We focus on the dentition of the pufferfish (Tetraodontidae) because of its distinct dental morphology. This complex dentition develops as a result of (i) a reduction in the number of tooth positions from seven to one per quadrant during the transition from first to second tooth generations and (ii) a dramatic shift in tooth morphogenesis following the development of the first-generation teeth, leading to the elongation of dental units along the jaw. Gene expression and 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) lineage tracing reveal a putative dental epithelial progenitor niche, suggesting a highly conserved mechanism for tooth regeneration despite the development of a unique dentition. MicroCT analysis reveals restricted labial openings in the beak, through which the dental epithelium (lamina) invades the cavity of the highly mineralized beak. Reduction in the number of replacement tooth positions coincides with the development of only four labial openings in the pufferfish beak, restricting connection of the oral epithelium to the dental cavity. Our data suggest the spatial restriction of dental regeneration, coupled with the unique extension of the replacement dental units throughout the jaw, are primary contributors to the evolution and development of this unique beak-like dentition.