The Impact of the Eda Pathway on Tooth Root Development.

The Eda pathway ( Eda, Edar, Edaradd) plays an important role in tooth development, determining tooth number, crown shape, and enamel formation. Here we show that the Eda pathway also plays a key role in root development. Edar (the receptor) is expressed in Hertwig's epithelial root sheath (HERS) during root development, with mutant mice showing a high incidence of taurodontism: large pulp chambers lacking or showing delayed bifurcation or trifurcation of the roots. The mouse upper second molars in the Eda pathway mutants show the highest incidence of taurodontism, this enhanced susceptibility being matched in human patients with mutations in EDA-A1. These taurodont teeth form due to defects in the direction of extension of the HERS from the crown, associated with a more extensive area of proliferation of the neighboring root mesenchyme. In those teeth where the angle at which the HERS extends from the crown is very wide and therefore more vertical, the mutant HERSs fail to reach toward the center of the tooth in the normal furcation region, and taurodont teeth are created. The phenotype is variable, however, with milder changes in angle and proliferation leading to normal or delayed furcation. This is the first analysis of the role of Eda in the root, showing a direct role for this pathway during postnatal mouse development, and it suggests that changes in proliferation and angle of HERS may underlie taurodontism in a range of syndromes.

Ectodysplasin A in Biological Fluids and Diagnosis of Ectodermal Dysplasia.

The tumor necrosis factor (TNF) family ligand ectodysplasin A (EDA) is produced as 2 full-length splice variants, EDA1 and EDA2, that bind to EDA receptor (EDAR) and X-linked EDA receptor (XEDAR/EDA2R), respectively. Inactivating mutations in Eda or Edar cause hypohidrotic ectodermal dysplasia (HED), a condition characterized by malformations of the teeth, hair and glands, with milder deficiencies affecting only the teeth. EDA acts early during the development of ectodermal appendages-as early as the embryonic placode stage-and plays a role in adult appendage function. In this study, the authors measured EDA in serum, saliva and dried blood spots. The authors detected 3- to 4-fold higher levels of circulating EDA in cord blood than in adult sera. A receptor binding-competent form of EDA1 was the main form of EDA but a minor fraction of EDA2 was also found in fetal bovine serum. Sera of EDA-deficient patients contained either background EDA levels or low levels of EDA that could not bind to recombinant EDAR. The serum of a patient with a V262F missense mutation in Eda, which caused a milder form of X-linked HED (XLHED), contained low levels of EDA capable of binding to EDAR. In 2 mildly affected carriers, intermediate levels of EDA were detected, whereas a severely affected carrier had no active EDA in the serum. Small amounts of EDA were also detectable in normal adult saliva. Finally, EDA could be measured in spots of wild-type adult or cord blood dried onto filter paper at levels significantly higher than that measured in EDA-deficient blood. Measurement of EDA levels combined with receptor-binding assays might be of relevance to aid in the diagnosis of total or partial EDA deficiencies.

Genetic factors controlling wool shedding in a composite Easycare sheep flock.

Historically, sheep have been selectively bred for desirable traits including wool characteristics. However, recent moves towards extensive farming and reduced farm labour have seen a renewed interest in Easycare breeds. The aim of this study was to quantify the underlying genetic architecture of wool shedding in an Easycare flock. Wool shedding scores were collected from 565 pedigreed commercial Easycare sheep from 2002 to 2010. The wool scoring system was based on a 10-point (0-9) scale, with score 0 for animals retaining full fleece and 9 for those completely shedding. DNA was sampled from 200 animals of which 48 with extreme phenotypes were genotyped using a 50-k SNP chip. Three genetic analyses were performed: heritability analysis, complex segregation analysis to test for a major gene hypothesis and a genome-wide association study to map regions in the genome affecting the trait. Phenotypes were treated as a continuous or binary variable and categories. High estimates of heritability (0.80 when treated as a continuous, 0.65-0.75 as binary and 0.75 as categories) for shedding were obtained from linear mixed model analyses. Complex segregation analysis gave similar estimates (0.80 ± 0.06) to those above with additional evidence for a major gene with dominance effects. Mixed model association analyses identified four significant (P < 0.05) SNPs. Further analyses of these four SNPs in all 200 animals revealed that one of the SNPs displayed dominance effects similar to those obtained from the complex segregation analyses. In summary, we found strong genetic control for wool shedding, demonstrated the possibility of a single putative dominant gene controlling this trait and identified four SNPs that may be in partial linkage disequilibrium with gene(s) controlling shedding.

Systems biology and livestock production.

The mapping of complete sets of genes, transcripts and proteins from many organisms has prompted the development of new '-omic' technologies for collecting and analysing very large amounts of data. Now that the tools to generate and interrogate such complete data sets are widely used, much of the focus of biological research has begun to turn towards understanding systems as a whole, rather than studying their components in isolation. This very broadly defined systems approach is being deployed across a range of problems and scales of organisation, including many aspects of the animal sciences. Here I review selected examples of this systems approach as applied to poultry and livestock production, product quality and welfare.

Computational and mathematical models of chondrogenesis in vertebrate limbs.

The production of cartilage (chondrogenic patterning) in the limb is one of the best-studied examples of the emergence of form in developmental biology. At the core of the theoretical study is an effort to understand the mechanism that establishes the characteristic distribution of cartilage in the embryonic limb, which defines the future sites and shapes of bones that will be present in the mature limb. This review article gives an overview of the history and current state of a rich literature of mathematical and computational models that seek to contribute to this problem. We describe models for the mechanisms of limb growth and shaping via interaction with various chemical fields, as well as models addressing the intrinsic self-organization capabilities of the embryonic mesenchymal tissue, such as reaction-diffusion and mechanochemical models. We discuss the contributions of these models to the current understanding of chondrogenesis in vertebrate limbs, as well as their relation to the varied conceptual models that have been proposed by experimentalists.

Towards an integrated experimental-theoretical approach for assessing the mechanistic basis of hair and feather morphogenesis.

In his seminal 1952 paper, 'The Chemical Basis of Morphogenesis', Alan Turing lays down a milestone in the application of theoretical approaches to understand complex biological processes. His deceptively simple demonstration that a system of reacting and diffusing chemicals could, under certain conditions, generate spatial patterning out of homogeneity provided an elegant solution to the problem of how one of nature's most intricate events occurs: the emergence of structure and form in the developing embryo. The molecular revolution that has taken place during the six decades following this landmark publication has now placed this generation of theoreticians and biologists in an excellent position to rigorously test the theory and, encouragingly, a number of systems have emerged that appear to conform to some of Turing's fundamental ideas. In this paper, we describe the history and more recent integration between experiment and theory in one of the key models for understanding pattern formation: the emergence of feathers and hair in the skins of birds and mammals.

Defects and rescue of the minor salivary glands in Eda pathway mutants.

Despite their importance to oral health, the mechanisms of minor salivary gland (SG) development are largely unexplored. Here we present in vivo and in vitro analyses of developing minor SGs in wild type and mutant mice. Eda, Shh and Fgf signalling pathway genes are expressed in these glands from an early stage of development. Developing minor SGs are absent in Eda pathway mutant embryos, and these mice exhibit a dysplastic circumvallate papilla with disrupted Shh expression. Supplementation of Eda pathway mutant minor SG explants with recombinant EDA rescues minor SG induction. Supplementation with Fgf8 or Shh, previously reported targets of Eda signalling, leads to induction of gland like structures in a few cases, but these fail to develop into minor SGs.

Recombinant EDA or Sonic Hedgehog rescue the branching defect in Ectodysplasin A pathway mutant salivary glands in vitro.

Hypohidrotic ectodermal dysplasia (HED) is characterized by defective ectodermal organ development. This includes the salivary glands (SGs), which have an important role in lubricating the oral cavity. In humans and mice, HED is caused by mutations in Ectodysplasin A (Eda) pathway genes. Various phenotypes of the mutant mouse Eda(Ta/Ta), which lacks the ligand Eda, can be rescued by maternal injection or in vitro culture supplementation with recombinant EDA. However, the response of the SGs to this treatment has not been investigated. Here, we show that the submandibular glands (SMGs) of Eda(Ta/Ta) mice exhibit impaired branching morphogenesis, and that supplementation of Eda(Ta/Ta) SMG explants with recombinant EDA rescues the defect. Supplementation of Edar(dlJ/dlJ) SMGs with recombinant Sonic hedgehog (Shh) also rescues the defect, whereas treatment with recombinant Fgf8 does not. This work is the first to test the ability of putative Eda target molecules to rescue Eda pathway mutant SMGs.

Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA.

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

A link between mRNA turnover and RNA interference in Arabidopsis.

In RNA interference (RNAi), double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA. In many organisms, RNA-dependent RNA polymerase (RdRp) is required to initiate or amplify RNAi, but the substrate for dsRNA synthesis in vivo is not known. Here, we show that RdRp-dependent transgene silencing in Arabidopsis was caused by mutation of XRN4, which is a ribonuclease (RNase) implicated in mRNA turnover by means of decapping and 5'-3' exonucleolysis. When both XRN4 and the RdRp were mutated, the plants accumulated decapped transgene mRNA. We propose that mRNAs lacking a cap structure become exposed to RdRp to initiate or maintain RNAi.

The activation level of the TNF family receptor, Edar, determines cusp number and tooth number during tooth development.

Mutations in members of the ectodysplasin (TNF-related) signalling pathway, EDA, EDAR, and EDARADD in mice and humans produce an ectodermal dysplasia phenotype that includes missing teeth and smaller teeth with reduced cusps. Using the keratin 14 promoter to target expression of an activated form of Edar in transgenic mice, we show that expression of this transgene is able to rescue the tooth phenotype in Tabby (Eda) and Sleek (Edar) mutant mice. High levels of expression of the transgene in wild-type mice result in molar teeth with extra cusps, and in some cases supernumerary teeth, the opposite of the mutant phenotype. The level of activation of Edar thus determines cusp number and tooth number during tooth development.

Construction of disturbed and intact soil blocks to develop percolating soil based treatment systems for dirty water from dairy farms.

Intact soil blocks with a surface area of 1.8 x 1.6 m, 1.0 m deep, were excavated in a coarse sandy loam. The sides of the soil blocks were supported with plywood before using hydraulic rams to force a steel cutting plate beneath them. Disturbed soil blocks of the same depth as the intact blocks were also established. Experiments were conducted to determine purification efficiencies for biological oxygen demand (BOD), molybdate reactive phosphorus (MRP), nitrate and ammonium-N after the application of dirty water. A preliminary experiment is described where a low application of dirty water was applied to the soil blocks, 2 mm day(-1). In addition, a chloride tracer was conducted for the duration of the experiment. Disturbed soil had a purification efficiency for BOD of 99% compared to 96% from intact soil (P<0.001). Purification efficiencies for MRP and ammonium-N were 100 and 99%, respectively, for the intact and disturbed soils. Nitrate-N concentration increased in leachate from both treatments reaching maximum concentrations of 15 and 8 mg l(-1) from disturbed and intact soils, respectively. Chloride traces for each soil block followed similar patterns with 47 and 51% loss from disturbed and intact soils, respectively.

Lactoferrin: influences on Langerhans cells, epidermal cytokines, and cutaneous inflammation.

It has been suggested previously that, in addition to other biological roles, lactoferrin (LF) may display antiinflammatory properties secondary to the regulation of cytokine expression. To explore this concept further, we have here examined in human volunteers the influence of recombinant homologous LF on the migration of epidermal Langerhans cells (LC), a process that is known to be dependent upon the local availability of certain proinflammatory cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). In common with previous studies in mice, it was found that topical administration of LF prior to exposure at the same site to the contact sensitizer diphenylcyclopropenone resulted in a significant reduction of allergen-induced LC migration from the epidermis (measured as a function of the frequency of CD1a+ or HLA-DR+ LC found in epidermal sheets prepared from punch biopsies of the treated skin sites). However, under the same conditions of exposure, LF was unable to influence migration of LC induced by the intradermal administration of TNF-alpha data consistent with the hypothesis that one action of LF in the skin is to regulate the local production of this cytokine. Further support for this hypothesis was derived from experiments conducted with IL-1beta. This cytokine is also able to induce the mobilization of LC following intradermal injection, although in this case, migration is known to be dependent upon the de novo production of TNF-alpha. We observed that prior exposure to LF resulted in a substantial inhibition of IL-1beta-induced LC migration, data again consistent with the regulation of TNF-alpha production by LF. Collectively, these results support the view that LF is able to influence cutaneous immune and inflammatory processes secondary to regulation of the production of TNF-alpha and possibly other cytokines.

Lactoferrin protects neonatal rats from gut-related systemic infection.

Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection. Proof for this concept is limited and was addressed during in vivo and in vitro studies. Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli. Control animals had 1,000-fold more colony-forming units of E. coli per milliliter of blood than treated animals (P < 0.001). Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02). Oral therapy with rh-LF + FeSO(4) did not alter the protective effect. In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages. An in vitro assay showed that rh-LF did not kill E. coli, but a combination of rh-LF + lysozyme was microbicidal. In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF. The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E. coli in vivo.

The winged helix/forkhead transcription factor Foxq1 regulates differentiation of hair in satin mice.

Satin (sa) homozygous mice have a silky coat with high sheen arising from structurally abnormal medulla cells and defects in differentiation of the hair shaft. We demonstrate that the winged helix/forkhead transcription factor, Foxq1 (Forkhead box, subclass q, member 1) is mutant in sa mice. An intragenic deletion was identified in the radiation-induced satin mutant of the SB/Le inbred strain; a second allele, identified by an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, has a missense mutation in the conserved winged helix DNA-binding domain. Homozygous mutants of the two alleles are indistinguishable. We show that Foxq1 is expressed during embryogenesis and exhibits a tissue-restricted expression pattern in adult tissues. The hair defects appear to be restricted to the inner structures of the hair; consequently, Foxq1 has a unique and distinct function involved in differentiation and development of the hair shaft. Despite an otherwise healthy appearance, satin mice have been reported to exhibit suppressed NK-cell function and alloimmune cytotoxic T-cell function. We show instead that the immune defects are attributable to genetic background differences.

Exogenous topical lactoferrin inhibits allergen-induced Langerhans cell migration and cutaneous inflammation in humans.

BACKGROUND: Lactoferrin (LF), an iron-binding protein found in exocrine secretions, is known to possess antibacterial properties. It has recently been proposed that LF may also influence inflammatory reactions. OBJECTIVES: To characterize in humans the ability of recombinant homologous LF to inhibit the induced migration of epidermal Langerhans cells (LCs) from the skin, a process known to be dependent upon the proinflammatory cytokines tumour necrosis factor (TNF)-alpha and interleukin 1beta and to influence cutaneous inflammatory reactions. METHODS: We investigated the anti-inflammatory properties of LF in human volunteers. RESULTS: Topical exposure to LF 2 h prior to sensitization caused a significant reduction in contact allergen (diphenylcyclopropenone, DPC)-induced LC migration from the epidermis as judged by the altered frequency of cells expressing either HLA-DR or CD1a determinants. That this reduction was secondary to an inhibition of TNF-alpha production was indicated by the fact that LF failed to influence LC migration induced by intradermal injection of this cytokine. In approximately 50% of those volunteers who displayed local inflammation in response to DPC, LF was found to cause a discernible reduction in the clinical severity of the reaction, associated with reduced infiltration of inflammatory cells. CONCLUSIONS: These data demonstrate that LF is able to influence cutaneous immune and inflammatory responses, possibly because of an impaired production of local proinflammatory cytokines.

X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling.

The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IkappaB kinase) complex, which is essential for NF-kappaB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

Gene defect in ectodermal dysplasia implicates a death domain adapter in development.

Members of the tumour-necrosis factor receptor (TNFR) family that contain an intracellular death domain initiate signalling by recruiting cytoplasmic death domain adapter proteins. Edar is a death domain protein of the TNFR family that is required for the development of hair, teeth and other ectodermal derivatives. Mutations in Edar-or its ligand, Eda-cause hypohidrotic ectodermal dysplasia in humans and mice. This disorder is characterized by sparse hair, a lack of sweat glands and malformation of teeth. Here we report the identification of a death domain adapter encoded by the mouse crinkled locus. The crinkled mutant has an hypohidrotic ectodermal dysplasia phenotype identical to that of the edar (downless) and eda (Tabby) mutants. This adapter, which we have called Edaradd (for Edar-associated death domain), interacts with the death domain of Edar and links the receptor to downstream signalling pathways. We also identify a missense mutation in its human orthologue, EDARADD, that is present in a family affected with hypohidrotic ectodermal dysplasia. Our findings show that the death receptor/adapter signalling mechanism is conserved in developmental, as well as apoptotic, signalling.

Edar/Eda interactions regulate enamel knot formation in tooth morphogenesis.

tabby and downless mutant mice have apparently identical defects in teeth, hair and sweat glands. Recently, genes responsible for these spontaneous mutations have been identified. downless (Dl) encodes Edar, a novel member of the tumour necrosis factor (TNF) receptor family, containing the characteristic extracellular cysteine rich fold, a single transmembrane region and a death homology domain close to the C terminus. tabby (Ta) encodes ectodysplasin-A (Eda) a type II membrane protein of the TNF ligand family containing an internal collagen-like domain. As predicted by the similarity in adult mutant phenotype and the structure of the proteins, we demonstrate that Eda and Edar specifically interact in vitro. We have compared the expression pattern of Dl and Ta in mouse development, taking the tooth as our model system, and find that they are not expressed in adjacent cells as would have been expected. Teeth develop by a well recorded series of epithelial-mesenchymal interactions, similar to those in hair follicle and sweat gland development, the structures found to be defective in tabby and downless mice. We have analysed the downless mutant teeth in detail, and have traced the defect in cusp morphology back to initial defects in the structure of the tooth enamel knot at E13. Significantly, the defect is distinct from that of the tabby mutant. In the tabby mutant, there is a recognisable but small enamel knot, whereas in the downless mutant the knot is absent, but enamel knot cells are organised into a different shape, the enamel rope, showing altered expression of signalling factors (Shh, Fgf4, Bmp4 and Wnt10b). By adding a soluble form of Edar to tooth germs, we were able to mimic the tabby enamel knot phenotype, demonstrating the involvement of endogenous Eda in tooth development. We could not, however, reproduce the downless phenotype, suggesting the existence of yet another ligand or receptor, or of ligand-independent activation mechanisms for Edar. Changes in the structure of the enamel knot signalling centre in downless tooth germs provide functional data directly linking the enamel knot with tooth cusp morphogenesis. We also show that the Lef1 pathway, thought to be involved in these mutants, functions independently in a parallel pathway.