Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans.

UNLABELLED: Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used "-like" suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. IMPORTANCE: The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens.

The effectiveness of beach mega-nourishment, assessed over three management epochs.

Resilient coastal protection requires adaptive management strategies that build with nature to maintain long-term sustainability. With increasing pressures on shorelines from urbanisation, industrial growth, sea-level rise and changing storm climates soft approaches to coastal management are implemented to support natural habitats and maintain healthy coastal ecosystems. The impact of a beach mega-nourishment along a frontage of interactive natural and engineered systems that incorporate soft and hard defences is explored. A coastal evolution model is applied to simulate the impact of different hypothetical mega-nourishment interventions to assess their impacts' over 3 shoreline management planning epochs: present-day (0-20 years), medium-term (20-50 years) and long-term (50-100 years). The impacts of the smaller interventions when appropriately positioned are found to be as effective as larger schemes, thus making them more cost-effective for present-day management. Over time the benefit from larger interventions becomes more noticeable, with multi-location schemes requiring a smaller initial nourishment to achieve at least the same benefit as that of a single-location scheme. While the longer-term impact of larger schemes reduces erosion across a frontage the short-term impact down drift of the scheme can lead to an increase in erosion as the natural sediment drift becomes interrupted. This research presents a transferable modelling tool to assess the impact of nourishment schemes for a variety of sedimentary shorelines and highlights both the positive and negative impact of beach mega-nourishment.

Fate and pathways of dredged estuarine sediment spoil in response to variable sediment size and baroclinic coastal circulation.

Most of the world's megacities are located in estuarine regions supporting commercial ports. Such locations are subject to sedimentation and require dredging to maintain activities. Liverpool Bay, northwest UK, is a region of freshwater influence and hypertidal conditions used to demonstrate the impact of baroclinicity when considering sediment disposal. Although tidal currents dominate the time-varying current and onshore sediment movement, baroclinic processes cause a 2-layer residual circulation that influences the longer-term sediment transport. A nested modelling system is applied to accurately simulate the circulation during a three month period. The hydrodynamic model is validated using coastal observations, and a Lagrangian particle tracking model is used to determine the pathways of 2 sediment mixtures representative of locally dredged material: a mix of 70% silt and 30% medium sand and a mix of 50% fine sand and 50% medium sand. Sediments are introduced at 3 active disposal sites within the Mersey Estuary in 2 different quantities (500 and 1500 Tonnes). Following release the majority (83% or more) of the particles remain within the estuary due to baroclinic influence. However, particles able to leave follow 2 distinct pathways, which primarily depend on the sediment grain size. Typically the finer sediment moves north and the coarser sediment west. Under solely barotropic conditions larger sediment volumes (up to 5 times more) can leave the estuary in a diffuse plume moving north. This demonstrates the necessity of considering baroclinic influence even within a hypertidal region with low freshwater inflow for accurate particle tracking studies.

Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison with bovine digital dermatitis treponemes.

This study aimed to isolate and characterize treponemes present in the bovine gastrointestinal (GI) tract and compare them with bovine digital dermatitis (BDD) treponemes. Seven spirochete isolates were obtained from the bovine GI tract, which, on the basis of 16S rRNA gene comparisons, clustered within the genus Treponema as four novel phylotypes. One phylotype was isolated from several different GI tract regions, including the omasum, colon, rumen, and rectum. These four phylotypes could be divided into two phylotype pairs that clustered closest with each other and then with different, previously reported rumen treponemes. The treponemes displayed great genotypic and phenotypic diversity between phylotypes and differed considerably from named treponeme species and those recently reported by metagenomic studies of the bovine GI tract. Phylogenetic inference, based on comparisons of 16S rRNA sequences from only bovine treponemes, suggested a marked divergence between two important groups. The dendrogram formed two major clusters, with one cluster containing GI tract treponemes and the other containing BDD treponemes. This division among the bovine treponemes is likely the result of adaptation to different niches. To further differentiate the bovine GI and BDD strains, we designed a degenerate PCR for a gene encoding a putative virulence factor, tlyC, which gave a positive reaction only for treponemes from the BDD cluster.

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry.

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.

Association of unique, isolated treponemes with bovine digital dermatitis lesions.

This study used a PCR-based approach targeting 16S rRNA gene fragments to determine the occurrence and association of the three bovine digital dermatitis (BDD) treponeme phylogroups within lesions found in cattle from the United Kingdom. Examination of 51 BDD lesions collected from infected cattle across the United Kingdom revealed that BDD treponeme group 1 (Treponema medium/Treponema vincentii-like), group 2 (Treponema phagedenis-like), and group 3 (Treponema putidum/Treponema denticola-like) were present in 96.1%, 98%, and 76.5% of BDD lesions, respectively. The three phylogroups were present together in 74.5% of lesions. The PCR assays enabled the isolation of further treponeme strains from previously mixed primary BDD lesion cultures. Here a representative from each of the three distinct treponeme phylogroups was isolated from a single BDD lesion for the first time. These data highlight the extent to which this disease is polytreponemal. Immunohistochemistry and electron microscopy were used to investigate lesional hoof tissues, resulting in treponemes being identified copiously in hair follicles and sebaceous glands, suggesting a potential route of exit and/or entry for these pathogens. This study gives further evidence for the importance of the three treponeme groups in BDD pathogenesis and reiterates the value of molecular genetic approaches for isolating and identifying fastidious anaerobes.

In vitro susceptibility of bovine digital dermatitis associated spirochaetes to antimicrobial agents.

Bovine digital dermatitis (BDD) is an infectious lameness in cattle, which has a large global impact in terms of animal welfare and cost. The majority of evidence suggests that spirochaetes are the aetiological agent of this disease. The aim of this study was to identify the susceptibility of BDD associated spirochaetes to a range of antimicrobial agents with a view to potential usage in vivo to treat this widespread cattle disease. A microdilution method was adapted to determine the in vitro susceptibilities of 19 UK digital dermatitis spirochaetes (6 Treponema medium/Treponema vincentii-like, 8 Treponema phagedenis-like and 5 Treponema denticola/Treponema putidum-like) to eight relevant antimicrobials. The BDD spirochaetes exhibited the highest susceptibility to penicillin and erythromycin and this information may now be used to aid development of efficacious treatments. This study has also identified that BDD spirochaete T167 is spectinomycin resistant and that the likely biological basis is a point mutation in the 16S rRNA gene. Interestingly, nearly all Brachyspira isolate 16S rRNA gene sequences in Genbank have this substitution, suggesting it may be responsible for the characteristic spectinomycin resistance reported for the Brachyspira genus.

Immunophenotypic expression of UCP1 in hibernoma and other adipose/non adipose soft tissue tumours.

BACKGROUND: Uncoupling protein 1 (UCP1) is a mitochondral protein transporter that uncouples electron transport from ATP production. UCP1 is highly expressed in brown adipose tissue (BAT), including hibernomas, but its expression in other adipose tumours is uncertain. UCP1 has also been found in other tissues (e.g. smooth muscle) but whether it is expressed in non-adipose benign and malignant soft tissue tumours is unknown. METHODS: Immunohistochemical staining of normal (axillary) BAT and subcutaneous/abdominal white adipose tissue (WAT) as well as a wide range of benign and malignant primary soft tissue tumours (n = 171) was performed using a rabbit polyclonal antibody to UCP1. BAT and hibernomas were also stained by immunohistochemistry with monoclonal and polyclonal antibodies to adipose/non-adipose tumour markers in order to characterise the immunophenotype of BAT cells. RESULTS: UCP1 was strongly expressed in the cytoplasm of brown fat cells in BAT and hibernomas, both of which also expressed aP2, S100, CD31, vimentin and calponin. UCP1 was not expressed in WAT or other adipose tumours with the exception a few tumour cells in pleomorphic liposarcoma. UCP1 was variably expressed by tumour cells in a few non-adipose sarcomas including leiomyosarcoma, rhabdomyosarcoma, alveolar soft part sarcoma, synovial sarcoma and clear cell sarcoma. CONCLUSIONS: UCP1 is strongly expressed in BAT but not WAT and is found in all hibernomas and a few pleomorphic liposarcomas but not in other adipose tumours. UCP1 expression in a few non-adipose soft tissue sarcomas may possibly reflect origin of tumour cells from a common mesenchymal stem cell precursor and/or developmental pathway.

Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle.

Bovine digital dermatitis (BDD) is a severe infectious cause of lameness which has spread through dairy cattle populations worldwide, causing serious welfare and agricultural problems. Spirochetes are the main organisms implicated and have previously proven difficult to isolate. This study aimed to isolate and characterise the range of spirochetes associated with BDD in the UK. Twenty-three spirochete isolates were obtained from 30 BDD lesions, which by 16S rRNA gene and flaB2 gene analysis clustered within the genus Treponema as three phylogroups; groups 1 (Treponema medium/Treponema vincentii-like), 2 (Treponema phagedenis-like) and 3 (Treponema denticola/Treponema putidum-like). The treponemes displayed large genotypic and phenotypic diversity between phylogroups and differed from named treponeme species. A previously isolated contagious ovine digital dermatitis spirochete was located within one of the three phylogroups, group 3, and could also be identified within this group on the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis may share the same aetiological agent. A strain isolated from a bovine interdigital dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine interdigital dermatitis and BDD may have the same causative agent. Two common enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD associated treponemes suggesting common metabolic pathways for sharing this novel niche or even common virulence traits. Further studies are required to determine whether the three groups of novel treponemes are representative of new treponeme taxa and to delineate how they interact with bovine tissues to cause disease.

Periostin expression in neoplastic and non-neoplastic diseases of bone and joint.

BACKGROUND: Periostin is a matricellular protein that is expressed in bone and joint tissues. To determine the expression of periostin in primary bone tumours and to assess whether it plays a role in tumour progression, we carried out immunohistochemistry and ELISA for periostin in a range of neoplastic and non-neoplastic bone and joint lesions. METHODS: 140 formalin-fixed paraffin-embedded sections of bone tumours and tumour-like lesions were stained by an indirect immunoperoxidase technique with a polyclonal anti-periostin antibody. Periostin expression was also assessed in rheumatoid arthritis (RA) and non-inflammatory osteoarthritis (OA) synovium and synovial fluid immunohistochemistry and ELISA respectively. RESULTS: Periostin was most strongly expressed in osteoid/woven bone of neoplastic and non-neoplastic bone-forming lesions, including osteoblastoma, osteosarcoma, fibrous dysplasia, osteofibrous dysplasia, fracture callus and myositis ossificans, and mineralised chondroid matrix/woven bone in chondroblastoma and clear cell chondrosarcoma. Reactive host bone at the edge of growing tumours, particularly in areas of increased vascularity and fibrosis, also stained strongly for periostin. Vascular elements in RA synovium strongly expressed periostin, and synovial fluid levels of periostin were higher in RA than OA. CONCLUSIONS: In keeping with its known role in modulating the synthesis of collagen and other extracellular matrix proteins in bone, strong periostin expression was noted in benign and malignant lesions forming an osteoid or osteoid-like matrix. Periostin was also noted in other bone tumours and was found in areas of reactive bone and increased vascularity at the edge of growing tumours, consistent with its involvement in tissue remodelling and angiogenesis associated with tumour progression.

An improved database of coastal flooding in the United Kingdom from 1915 to 2016.

Coastal flooding caused by extreme sea levels can produce devastating and wide-ranging consequences. The 'SurgeWatch' v1.0 database systematically documents and assesses the consequences of historical coastal flood events around the UK. The original database was inevitably biased due to the inconsistent spatial and temporal coverage of sea-level observations utilised. Therefore, we present an improved version integrating a variety of 'soft' data such as journal papers, newspapers, weather reports, and social media. SurgeWatch2.0 identifies 329 coastal flooding events from 1915 to 2016, a more than fivefold increase compared to the 59 events in v1.0. Moreover, each flood event is now ranked using a multi-level categorisation based on inundation, transport disruption, costs, and fatalities: from 1 (Nuisance) to 6 (Disaster). For the 53 most severe events ranked Category 3 and above, an accompanying event description based upon the Source-Pathway-Receptor-Consequence framework was produced. Thus, SurgeWatch v2.0 provides the most comprehensive and coherent historical record of UK coastal flooding. It is designed to be a resource for research, planning, management and education.

Spatial and temporal analysis of extreme sea level and storm surge events around the coastline of the UK.

In this paper we analyse the spatial footprint and temporal clustering of extreme sea level and skew surge events around the UK coast over the last 100 years (1915-2014). The vast majority of the extreme sea level events are generated by moderate, rather than extreme skew surges, combined with spring astronomical high tides. We distinguish four broad categories of spatial footprints of events and the distinct storm tracks that generated them. There have been rare events when extreme levels have occurred along two unconnected coastal regions during the same storm. The events that occur in closest succession (<4 days) typically impact different stretches of coastline. The spring/neap tidal cycle prevents successive extreme sea level events from happening within 4-8 days. Finally, the 2013/14 season was highly unusual in the context of the last 100 years from an extreme sea level perspective.

Physical and economic impacts of sea-level rise and low probability flooding events on coastal communities.

Conventionally flood mapping typically includes only a static water level (e.g. peak of a storm tide) in coastal flood inundation events. Additional factors become increasingly important when increased water-level thresholds are met during the combination of a storm tide and increased mean sea level. This research incorporates factors such as wave overtopping and river flow in a range of flood inundation scenarios of future sea-level projections for a UK case study of Fleetwood, northwest England. With increasing mean sea level it is shown that wave overtopping and river forcing have an important bearing on the cost of coastal flood events. The method presented converts inundation maps into monetary cost. This research demonstrates that under scenarios of joint extreme surge-wave-river events the cost of flooding can be increased by up to a factor of 8 compared with an increase in extent of up to a factor of 3 relative to "surge alone" event. This is due to different areas being exposed to different flood hazards and areas with common hazard where flood waters combine non-linearly. This shows that relying simply on flood extent and volume can under-predict the actual economic impact felt by a coastal community. Additionally, the scenario inundation depths have been presented as "brick course" maps, which represent a new way of interpreting flood maps. This is primarily aimed at stakeholders to increase levels of engagement within the coastal community.

A user-friendly database of coastal flooding in the United Kingdom from 1915-2014.

Coastal flooding caused by extreme sea levels can be devastating, with long-lasting and diverse consequences. Historically, the UK has suffered major flooding events, and at present 2.5 million properties and £150 billion of assets are potentially exposed to coastal flooding. However, no formal system is in place to catalogue which storms and high sea level events progress to coastal flooding. Furthermore, information on the extent of flooding and associated damages is not systematically documented nationwide. Here we present a database and online tool called 'SurgeWatch', which provides a systematic UK-wide record of high sea level and coastal flood events over the last 100 years (1915-2014). Using records from the National Tide Gauge Network, with a dataset of exceedance probabilities and meteorological fields, SurgeWatch captures information of 96 storms during this period, the highest sea levels they produced, and the occurrence and severity of coastal flooding. The data are presented to be easily assessable and understandable to a range of users including, scientists, coastal engineers, managers and planners and concerned citizens.

Lack of benefit of intravenous synthetic human secretin in the treatment of autism.

The objective of this study was to determine if an intravenous infusion of synthetic human secretin improves language and behavioral symptoms in children with autism. Forty-two children with the diagnosis of autism were randomized to one of two groups in this double-blind cross-over trial. One group received 2 IU/kg of intravenous synthetic human secretin at the first visit, followed by an equal volume of intravenous saline placebo at week 6. The other group received treatments in the reverse order. All children were evaluated at weeks 1, 3, 6, 9, and 12 with standardized assessments of language, behavior, and autism symptomatology. There were no significant differences in the mean scores on any measure of language, behavior, or autism symptom severity after treatment with secretin compared to treatment with placebo. The results of this study do not support secretin as a treatment for autism.

Differential inflammatory responses of bovine foot skin fibroblasts and keratinocytes to digital dermatitis treponemes.

Bovine digital dermatitis (BDD) is a serious infectious inflammatory lameness causing pain and suffering to many cattle worldwide and which has severe economic implications. This study set out to investigate relationships between the treponemes considered causal of BDD and the local inflammatory response of the bovine host. Here we describe, for the first time, the isolation of bovine foot skin keratinocytes and fibroblasts as separate cell lineages. These cell lines were then exposed to treponeme whole-cell sonicates, and the gene expression of selected host inflammatory mediators investigated using quantitative reverse transcriptase PCR. Several genes, including those encoding RANTES/CCL5, MMP12, TNFα, TGFß and TIMP3 were significantly upregulated in fibroblasts exposed to whole-cell sonicates derived from BDD treponeme phylotypes. For each of the above genes there were similar fibroblast expression increases for all three BDD treponeme phylotypes tested, suggesting common virulence mechanisms. With bovine foot skin keratinocytes, we were unable to detect expression of RANTES/CCL5 and after incubation with BDD treponeme constituents we were unable to observe any significant changes in expression of inflammatory mediators tested. These contrasting results suggest fibroblasts rather than keratinocytes may be an important shared target of pathogenesis for BDD treponemes.

Host and environmental reservoirs of infection for bovine digital dermatitis treponemes.

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.

Treponema pedis sp. nov., a spirochaete isolated from bovine digital dermatitis lesions.

Bovine digital dermatitis (BDD) is a debilitating infection that is being increasingly recognized in livestock worldwide. Several treponeme phylotypes have been identified in BDD lesions, although only a single BDD-associated treponeme taxon (Treponema brennaborense) has been proposed thus far. In a previous study, we observed that four BDD-associated spirochaete isolates formed a distinct phylogenetic cluster on the basis of 16S rRNA gene sequence analysis and shared less than 97 % sequence similarity with recognized treponeme species. Further characterization of these isolates on the basis of growth characteristics, flaB2 gene sequences, enzyme profiles and cell morphology confirmed that they formed a coherent taxonomic group displaying marked genotypic and phenotypic differences with respect to recognized treponeme species. The four novel isolates displayed a novel 3 : 6 : 3 flagellar pattern rather than the 2 : 4 : 2 pattern shown by their closest relatives and exhibited esterase C4, esterase lipase C8, trypsin and chymotrypsin enzyme activities. Therefore these four new isolates represent a novel species of the genus Treponema, for which the name Treponema pedis sp. nov. is proposed. The type strain is T3552B(T) (=DSM 18691(T)=NCTC 13403(T)).

Unilateral nerve injury produces bilateral loss of distal innervation.

There are no known anatomical connections between neurons that innervate homologous right and left body parts. Nevertheless, some patients develop bilateral abnormalities after unilateral injury, a phenomenon often unrecognized and not yet characterized. Therefore, we examined in rats the effects of ligating and cutting one tibial nerve on sensory function and on density of innervation in hind paws contralaterally as well as ipsilaterally to the injury, at times between 1 day and 5 months after surgery. Punches removed from tibial- or sural-innervated planter paw skin were immunolabeled to quantitate epidermal nerve endings. Naive and sham-operated rats provided controls. Axotomized rats had near-total loss of PGP9.5(+) innervation within ipsilateral tibial-innervated skin at all time-points. Adjacent ipsilateral sural-innervated skin had persistent hyperalgesia without denervation, and robust axonal sprouting at 5 months after surgery. Contralesional hind paws lost 54% of innervation in tibial-innervated epidermis starting 1 week after surgery and persisting throughout. Contralesional sural-innervated skin had neither neurite loss nor sprouting. These results imply that unilateral nerve injury can cause profound, long lasting, nerve-branch-specific loss of distal innervation contralaterally as well as ipsilaterally. They discredit the practice of using tissues contralateral to an injury to provide normative controls and suggest the possibility of rapid, transmedian postinjury signals between homologous mirror-image neurons.

Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot.

The pre-axial polydactylous mouse mutant Doublefoot has 6-9 digits per limb but lacks anteroposterior polarity (there is no biphalangeal digit 1). It differs from other polydactylous mutants in showing normal Shh expression, but polarizing activity (shown by mouse-chick grafting experiments) and hedgehog signalling activity (shown by expression of Ptc1) are present throughout the distal mesenchyme. The Dbf mutation has not yet been identified. Here we review current understanding of this mutant, and briefly report new results indicating (1) that limb bud expansion is concomitant with ectopic lhh expression and with extension of the posterior high cell proliferation rate into the anterior region, and (2) that the Dbf mutation is epistatic to Shh in the limb.