Novel Aspects in Pattern Formation Arise from Coupling Turing Reaction-Diffusion and Chemotaxis.

Recent experimental studies on primary hair follicle formation and feather bud morphogenesis indicate a coupling between Turing-type diffusion driven instability and chemotactic patterning. Inspired by these findings we develop and analyse a mathematical model that couples chemotaxis to a reaction-diffusion system exhibiting diffusion-driven (Turing) instability. While both systems, reaction-diffusion systems and chemotaxis, can independently generate spatial patterns, we were interested in how the coupling impacts the stability of the system, parameter region for patterning, pattern geometry, as well as the dynamics of pattern formation. We conduct a classical linear stability analysis for different model structures, and confirm our results by numerical analysis of the system. Our results show that the coupling generally increases the robustness of the patterning process by enlarging the pattern region in the parameter space. Concerning time scale and pattern regularity, we find that an increase in the chemosensitivity can speed up the patterning process for parameters inside and outside of the Turing space, but generally reduces spatial regularity of the pattern. Interestingly, our analysis indicates that pattern formation can also occur when neither the Turing nor the chemotaxis system can independently generate pattern. On the other hand, for some parameter settings, the coupling of the two processes can extinguish the pattern formation, rather than reinforce it. These theoretical findings can be used to corroborate the biological findings on morphogenesis and guide future experimental studies. From a mathematical point of view, this work sheds a light on coupling classical pattern formation systems from the parameter space perspective.

Rapid mechanosensitive migration and dispersal of newly divided mesenchymal cells aid their recruitment into dermal condensates.

Embryonic mesenchymal cells are dispersed within an extracellular matrix but can coalesce to form condensates with key developmental roles. Cells within condensates undergo fate and morphological changes and induce cell fate changes in nearby epithelia to produce structures including hair follicles, feathers, or intestinal villi. Here, by imaging mouse and chicken embryonic skin, we find that mesenchymal cells undergo much of their dispersal in early interphase, in a stereotyped process of displacement driven by 3 hours of rapid and persistent migration followed by a long period of low motility. The cell division plane and the elevated migration speed and persistence of newly born mesenchymal cells are mechanosensitive, aligning with tissue tension, and are reliant on active WNT secretion. This behaviour disperses mesenchymal cells and allows daughters of recent divisions to travel long distances to enter dermal condensates, demonstrating an unanticipated effect of cell cycle subphase on core mesenchymal behaviour.

The developmental basis of fingerprint pattern formation and variation.

Fingerprints are complex and individually unique patterns in the skin. Established prenatally, the molecular and cellular mechanisms that guide fingerprint ridge formation and their intricate arrangements are unknown. Here we show that fingerprint ridges are epithelial structures that undergo a truncated hair follicle developmental program and fail to recruit a mesenchymal condensate. Their spatial pattern is established by a Turing reaction-diffusion system, based on signaling between EDAR, WNT, and antagonistic BMP pathways. These signals resolve epithelial growth into bands of focalized proliferation under a precociously differentiated suprabasal layer. Ridge formation occurs as a set of waves spreading from variable initiation sites defined by the local signaling environments and anatomical intricacies of the digit, with the propagation and meeting of these waves determining the type of pattern that forms. Relying on a dynamic patterning system triggered at spatially distinct sites generates the characteristic types and unending variation of human fingerprint patterns.

The EDA-deficient mouse has Zymbal's gland hypoplasia and acute otitis externa.

In mice, rats, dogs and humans, the growth and function of sebaceous glands and eyelid Meibomian glands depend on the ectodysplasin signalling pathway. Mutation of genes encoding the ligand EDA, its transmembrane receptor EDAR and the intracellular signal transducer EDARADD leads to hypohidrotic ectodermal dysplasia, characterised by impaired development of teeth and hair, as well as cutaneous glands. The rodent ear canal has a large auditory sebaceous gland, the Zymbal's gland, the function of which in the health of the ear canal has not been determined. We report that EDA-deficient mice, EDAR-deficient mice and EDARADD-deficient rats have Zymbal's gland hypoplasia. EdaTa mice have 25% prevalence of otitis externa at postnatal day 21 and treatment with agonist anti-EDAR antibodies rescues Zymbal's glands. The aetiopathogenesis of otitis externa involves infection with Gram-positive cocci, and dosing pregnant and lactating EdaTa females and pups with enrofloxacin reduces the prevalence of otitis externa. We infer that the deficit of sebum is the principal factor in predisposition to bacterial infection, and the EdaTa mouse is a potentially useful microbial challenge model for human acute otitis externa.

Limb development genes underlie variation in human fingerprint patterns.

Fingerprints are of long-standing practical and cultural interest, but little is known about the mechanisms that underlie their variation. Using genome-wide scans in Han Chinese cohorts, we identified 18 loci associated with fingerprint type across the digits, including a genetic basis for the long-recognized "pattern-block" correlations among the middle three digits. In particular, we identified a variant near EVI1 that alters regulatory activity and established a role for EVI1 in dermatoglyph patterning in mice. Dynamic EVI1 expression during human development supports its role in shaping the limbs and digits, rather than influencing skin patterning directly. Trans-ethnic meta-analysis identified 43 fingerprint-associated loci, with nearby genes being strongly enriched for general limb development pathways. We also found that fingerprint patterns were genetically correlated with hand proportions. Taken together, these findings support the key role of limb development genes in influencing the outcome of fingerprint patterning.

The Enrichment of Specific Hair Follicle-Associated Cell Populations in Plucked Hairs Offers an Opportunity to Study Gene Expression Underlying Hair Traits.

Gene expression differences can assist in characterizing important underlying genetic mechanisms between different phenotypic traits. However, when population-dense tissues are studied, the signals from scarce populations are diluted. Therefore, appropriately choosing a sample collection method that enriches a particular type of effector cells might yield more specific results. To address this issue, we performed a polyA-selected RNA-seq experiment of domestic horse (Equus ferus caballus) plucked-hair samples and skin biopsies. Then, we layered the horse gene abundance results against cell type-specific marker genes generated from a scRNA-seq supported with spatial mapping of laboratory mouse (Mus musculus) skin to identify the captured populations. The hair-plucking and skin-biopsy sample-collection methods yielded comparable quality and quantity of RNA-seq results. Keratin-related genes, such as KRT84 and KRT75, were among the genes that showed higher abundance in plucked hairs, while genes involved in cellular processes and enzymatic activities, such as MGST1, had higher abundance in skin biopsies. We found an enrichment of hair-follicle keratinocytes in plucked hairs, but detected an enrichment of other populations, including epidermis keratinocytes, in skin biopsies. In mammalian models, biopsies are often the method of choice for a plethora of gene expression studies and to our knowledge, this is a novel study that compares the cell-type enrichment between the non-invasive hair-plucking and the invasive skin-biopsy sample-collection methods. Here, we show that the non-invasive and ethically uncontroversial plucked-hair method is recommended depending on the research question. In conclusion, our study will allow downstream -omics approaches to better understand integumentary conditions in both health and disease in horses as well as other mammals.

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia.

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong ß-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes ß-catenin. Deletion of this exon yields unconstrained ß-catenin signalling activity. We also demonstrate that ß-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which ß-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.

Systems for intricate patterning of the vertebrate anatomy.

Periodic patterns form intricate arrays in the vertebrate anatomy, notably the hair and feather follicles of the skin, but also internally the villi of the gut and the many branches of the lung, kidney, mammary and salivary glands. These tissues are composite structures, being composed of adjoined epithelium and mesenchyme, and the patterns that arise within them require interaction between these two tissue layers. In embryonic development, cells change both their distribution and state in a periodic manner, defining the size and relative positions of these specialized structures. Their placement is determined by simple spacing mechanisms, with substantial evidence pointing to a variety of local enhancement/lateral inhibition systems underlying the breaking of symmetry. The nature of the cellular processes involved, however, has been less clear. While much attention has focused on intercellular soluble signals, such as protein growth factors, experimental evidence has grown for contributions of cell movement or mechanical forces to symmetry breaking. In the mesenchyme, unlike the epithelium, cells may move freely and can self-organize into aggregates by chemotaxis, or through generation and response to mechanical strain on their surrounding matrix. Different modes of self-organization may coexist, either coordinated into a single system or with hierarchical relationships. Consideration of a broad range of distinct biological processes is required to advance understanding of biological pattern formation. This article is part of the theme issue 'Recent progress and open frontiers in Turing's theory of morphogenesis'.

A genome-wide association analysis for body weight at 35 days measured on 137,343 broiler chickens.

BACKGROUND: Body weight (BW) is an economically important trait in the broiler (meat-type chickens) industry. Under the assumption of polygenicity, a "large" number of genes with "small" effects is expected to control BW. To detect such effects, a large sample size is required in genome-wide association studies (GWAS). Our objective was to conduct a GWAS for BW measured at 35 days of age with a large sample size. METHODS: The GWAS included 137,343 broilers spanning 15 pedigree generations and 392,295 imputed single nucleotide polymorphisms (SNPs). A false discovery rate of 1% was adopted to account for multiple testing when declaring significant SNPs. A Bayesian ridge regression model was implemented, using AlphaBayes, to estimate the contribution to the total genetic variance of each region harbouring significant SNPs (1 Mb up/downstream) and the combined regions harbouring non-significant SNPs. RESULTS: GWAS revealed 25 genomic regions harbouring 96 significant SNPs on 13 Gallus gallus autosomes (GGA1 to 4, 8, 10 to 15, 19 and 27), with the strongest associations on GGA4 at 65.67-66.31 Mb (Galgal4 assembly). The association of these regions points to several strong candidate genes including: (i) growth factors (GGA1, 4, 8, 13 and 14); (ii) leptin receptor overlapping transcript (LEPROT)/leptin receptor (LEPR) locus (GGA8), and the STAT3/STAT5B locus (GGA27), in connection with the JAK/STAT signalling pathway; (iii) T-box gene (TBX3/TBX5) on GGA15 and CHST11 (GGA1), which are both related to heart/skeleton development); and (iv) PLAG1 (GGA2). Combined together, these 25 genomic regions explained ~ 30% of the total genetic variance. The region harbouring significant SNPs that explained the largest portion of the total genetic variance (4.37%) was on GGA4 (~ 65.67-66.31 Mb). CONCLUSIONS: To the best of our knowledge, this is the largest GWAS that has been conducted for BW in chicken to date. In spite of the identified regions, which showed a strong association with BW, the high proportion of genetic variance attributed to regions harbouring non-significant SNPs supports the hypothesis that the genetic architecture of BW35 is polygenic and complex. Our results also suggest that a large sample size will be required for future GWAS of BW35.

Isolating Patterns in Open Reaction-Diffusion Systems.

Realistic examples of reaction-diffusion phenomena governing spatial and spatiotemporal pattern formation are rarely isolated systems, either chemically or thermodynamically. However, even formulations of 'open' reaction-diffusion systems often neglect the role of domain boundaries. Most idealizations of closed reaction-diffusion systems employ no-flux boundary conditions, and often patterns will form up to, or along, these boundaries. Motivated by boundaries of patterning fields related to the emergence of spatial form in embryonic development, we propose a set of mixed boundary conditions for a two-species reaction-diffusion system which forms inhomogeneous solutions away from the boundary of the domain for a variety of different reaction kinetics, with a prescribed uniform state near the boundary. We show that these boundary conditions can be derived from a larger heterogeneous field, indicating that these conditions can arise naturally if cell signalling or other properties of the medium vary in space. We explain the basic mechanisms behind this pattern localization and demonstrate that it can capture a large range of localized patterning in one, two, and three dimensions and that this framework can be applied to systems involving more than two species. Furthermore, the boundary conditions proposed lead to more symmetrical patterns on the interior of the domain and plausibly capture more realistic boundaries in developmental systems. Finally, we show that these isolated patterns are more robust to fluctuations in initial conditions and that they allow intriguing possibilities of pattern selection via geometry, distinct from known selection mechanisms.

Methods for the Administration of EDAR Pathway Modulators in Mice.

Genetic deficiency of ectodysplasin A (EDA) causes X-linked hypohidrotic ectodermal dysplasia, a congenital condition characterized by the absence or abnormal formation of sweat glands, teeth, and several skin appendages. Stimulation of the EDA receptor (EDAR) with agonists in the form of recombinant EDA or anti-EDAR antibodies can compensate for the absence of Eda in a mouse model of Eda deficiency, provided that agonists are administered in a timely manner during fetal development. Here we provide detailed protocols for the administration of EDAR agonists or antagonists, or other proteins, by the intravenous, intraperitoneal, and intra-amniotic routes as well as protocols to collect blood, to visualize sweat gland function, and to prepare skulls in mice.

Characterisation of a second gain of function EDAR variant, encoding EDAR380R, in East Asia.

Ectodysplasin A1 receptor (EDAR) is a TNF receptor family member with roles in the development and growth of hair, teeth and glands. A derived allele of EDAR, single-nucleotide variant rs3827760, encodes EDAR:p.(Val370Ala), a receptor with more potent signalling effects than the ancestral EDAR370Val. This allele of rs3827760 is at very high frequency in modern East Asian and Native American populations as a result of ancient positive selection and has been associated with straighter, thicker hair fibres, alteration of tooth and ear shape, reduced chin protrusion and increased fingertip sweat gland density. Here we report the characterisation of another SNV in EDAR, rs146567337, encoding EDAR:p.(Ser380Arg). The derived allele of this SNV is at its highest global frequency, of up to 5%, in populations of southern China, Vietnam, the Philippines, Malaysia and Indonesia. Using haplotype analyses, we find that the rs3827760 and rs146567337 SNVs arose on distinct haplotypes and that rs146567337 does not show the same signs of positive selection as rs3827760. From functional studies in cultured cells, we find that EDAR:p.(Ser380Arg) displays increased EDAR signalling output, at a similar level to that of EDAR:p.(Val370Ala). The existence of a second SNV with partly overlapping geographic distribution, the same in vitro functional effect and similar evolutionary age as the derived allele of rs3827760, but of independent origin and not exhibiting the same signs of strong selection, suggests a northern focus of positive selection on EDAR function in East Asia.

Role of ectodysplasin signalling in middle ear and nasal pathology in rat and mouse models of hypohidrotic ectodermal dysplasia.

Patients with mutations in the ectodysplasin receptor signalling pathway genes - the X-linked ligand ectodysplasin-A (EDA), the receptor EDAR or the receptor adapter EDARADD - have hypohidrotic ectodermal dysplasia (HED). In addition to having impaired development of teeth, hair, eccrine sweat glands, and salivary and mammary glands, HED patients have ear, nose and throat disease. The mouse strains Tabby (EdaTa ) and downless (Edardl-J/dl-J ) have rhinitis and otitis media due to loss of submucosal glands in the upper airway. We report that prenatal correction of EDAR signalling in EdaTa mice with the agonist anti-EDAR antibody rescues the auditory-tube submucosal glands and prevents otitis media, rhinitis and nasopharyngitis. The sparse- and wavy-haired (swh) rat strain carries a mutation in the Edaradd gene and has similar cutaneous HED phenotypes to mouse models. We report that auditory-tube submucosal glands are smaller in the homozygous mutant Edaraddswh/swh than those in unaffected heterozygous Edaraddswh/+ rats, and that this predisposes them to otitis media. Furthermore, the pathogenesis of otitis media in the rat HED model differs from that in mice, as otitis media is the primary pathology, and rhinitis is a later-onset phenotype. These findings in rodent HED models imply that hypomorphic as well as null mutations in EDAR signalling pathway genes may predispose to otitis media in humans. In addition, this work suggests that the recent successful prenatal treatment of X-linked HED (XLHED) in humans may also prevent ear, nose and throat disease, and provides diagnostic criteria that distinguish HED-associated otitis media from chronic otitis media with effusion, which is common in children.

Feather arrays are patterned by interacting signalling and cell density waves.

Feathers are arranged in a precise pattern in avian skin. They first arise during development in a row along the dorsal midline, with rows of new feather buds added sequentially in a spreading wave. We show that the patterning of feathers relies on coupled fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling together with mesenchymal cell movement, acting in a coordinated reaction-diffusion-taxis system. This periodic patterning system is partly mechanochemical, with mechanical-chemical integration occurring through a positive feedback loop centred on FGF20, which induces cell aggregation, mechanically compressing the epidermis to rapidly intensify FGF20 expression. The travelling wave of feather formation is imposed by expanding expression of Ectodysplasin A (EDA), which initiates the expression of FGF20. The EDA wave spreads across a mesenchymal cell density gradient, triggering pattern formation by lowering the threshold of mesenchymal cells required to begin to form a feather bud. These waves, and the precise arrangement of feather primordia, are lost in the flightless emu and ostrich, though via different developmental routes. The ostrich retains the tract arrangement characteristic of birds in general but lays down feather primordia without a wave, akin to the process of hair follicle formation in mammalian embryos. The embryonic emu skin lacks sufficient cells to enact feather formation, causing failure of tract formation, and instead the entire skin gains feather primordia through a later process. This work shows that a reaction-diffusion-taxis system, integrated with mechanical processes, generates the feather array. In flighted birds, the key role of the EDA/Ectodysplasin A receptor (EDAR) pathway in vertebrate skin patterning has been recast to activate this process in a quasi-1-dimensional manner, imposing highly ordered pattern formation.

Edar is a downstream target of beta-catenin and drives collagen accumulation in the mouse prostate.

Beta-catenin (CTNNB1) directs ectodermal appendage spacing by activating ectodysplasin A receptor (EDAR) transcription, but whether CTNNB1 acts by a similar mechanism in the prostate, an endoderm-derived tissue, is unclear. Here we examined the expression, function, and CTNNB1 dependence of the EDAR pathway during prostate development. In situ hybridization studies reveal EDAR pathway components including Wnt10b in the developing prostate and localize these factors to prostatic bud epithelium where CTNNB1 target genes are co-expressed. We used a genetic approach to ectopically activate CTNNB1 in developing mouse prostate and observed focal increases in Edar and Wnt10b mRNAs. We also used a genetic approach to test the prostatic consequences of activating or inhibiting Edar expression. Edar overexpression does not visibly alter prostatic bud formation or branching morphogenesis, and Edar expression is not necessary for either of these events. However, Edar overexpression is associated with an abnormally thick and collagen-rich stroma in adult mouse prostates. These results support CTNNB1 as a transcriptional activator of Edar and Wnt10b in the developing prostate and demonstrate Edar is not only important for ectodermal appendage patterning but also influences collagen organization in adult prostates.This article has an associated First Person interview with the first author of the paper.

A chemotaxis model of feather primordia pattern formation during avian development.

The orderly formation of the avian feather array is a classic example of periodic pattern formation during embryonic development. Various mathematical models have been developed to describe this process, including Turing/activator-inhibitor type reaction-diffusion systems and chemotaxis/mechanical-based models based on cell movement and tissue interactions. In this paper we formulate a mathematical model founded on experimental findings, a set of interactions between the key cellular (dermal and epidermal cell populations) and molecular (fibroblast growth factor, FGF, and bone morphogenetic protein, BMP) players and a medially progressing priming wave that acts as the trigger to initiate patterning. Linear stability analysis is used to show that FGF-mediated chemotaxis of dermal cells is the crucial driver of pattern formation, while perturbations in the form of ubiquitous high BMP expression suppress patterning, consistent with experiments. Numerical simulations demonstrate the capacity of the model to pattern the skin in a spatial-temporal manner analogous to avian feather development. Further, experimental perturbations in the form of bead-displacement experiments are recapitulated and predictions are proposed in the form of blocking mesenchymal cell proliferation.

Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas.

Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway. Although most cases of BCC are sporadic, some forms are inherited, such as Bazex-Dupré-Christol syndrome (BDCS)-a cancer-prone genodermatosis with an X-linked, dominant inheritance pattern. We have identified mutations in the ACTRT1 gene, which encodes actin-related protein T1 (ARP-T1), in two of the six families with BDCS that were examined in this study. High-throughput sequencing in the four remaining families identified germline mutations in noncoding sequences surrounding ACTRT1. These mutations were located in transcribed sequences encoding enhancer RNAs (eRNAs) and were shown to impair enhancer activity and ACTRT1 expression. ARP-T1 was found to directly bind to the GLI1 promoter, thus inhibiting GLI1 expression, and loss of ARP-T1 led to activation of the Hedgehog pathway in individuals with BDCS. Moreover, exogenous expression of ACTRT1 reduced the in vitro and in vivo proliferation rates of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCC involving mutations in regulatory noncoding elements and uncovers the tumor-suppressor properties of ACTRT1.

Hierarchical patterning modes orchestrate hair follicle morphogenesis.

Two theories address the origin of repeating patterns, such as hair follicles, limb digits, and intestinal villi, during development. The Turing reaction-diffusion system posits that interacting diffusible signals produced by static cells first define a prepattern that then induces cell rearrangements to produce an anatomical structure. The second theory, that of mesenchymal self-organisation, proposes that mobile cells can form periodic patterns of cell aggregates directly, without reference to any prepattern. Early hair follicle development is characterised by the rapid appearance of periodic arrangements of altered gene expression in the epidermis and prominent clustering of the adjacent dermal mesenchymal cells. We assess the contributions and interplay between reaction-diffusion and mesenchymal self-organisation processes in hair follicle patterning, identifying a network of fibroblast growth factor (FGF), wingless-related integration site (WNT), and bone morphogenetic protein (BMP) signalling interactions capable of spontaneously producing a periodic pattern. Using time-lapse imaging, we find that mesenchymal cell condensation at hair follicles is locally directed by an epidermal prepattern. However, imposing this prepattern's condition of high FGF and low BMP activity across the entire skin reveals a latent dermal capacity to undergo spatially patterned self-organisation in the absence of epithelial direction. This mesenchymal self-organisation relies on restricted transforming growth factor (TGF) ß signalling, which serves to drive chemotactic mesenchymal patterning when reaction-diffusion patterning is suppressed, but, in normal conditions, facilitates cell movement to locally prepatterned sources of FGF. This work illustrates a hierarchy of periodic patterning modes operating in organogenesis.

Reversing stratification during wound healing.

The involvement of proliferation and migration in epidermal healing has long been recognized, but three studies now reveal how a variety of individual cell behaviours achieve a collective epithelial response, and how diverse repair routes are taken by cells of different origins.