Integrating orientation mechanisms, adrenocortical activity, and endurance flight in vagrancy behaviour.

Avian migratory processes are typically precisely oriented, yet vagrants are frequently recorded outside their normal range. Wind displaced vagrants often show corrective behaviour, and as an appropriate response is likely adaptive. We investigated the physiological response to vagrancy in passerines. Activation of the emergency life-history stage (ELHS), assessed by high baseline plasma corticosterone, is a potential mechanism to elicit compensatory behaviour in response to challenges resulting from navigational error, coupled with response to fuel load and flight. We compared circulating plasma corticosterone concentrations and body condition between three migratory groups in autumn: (1) wind displaced southwest (SW) vagrants and (2) long range southeast (SE) vagrants on the remote Faroe Islands, and (3) birds within the expected SW migratory route (controls) on the Falsterbo peninsula, Sweden. Vagrants were further grouped by those sampled immediately upon termination of over-water migratory flight and those already on the island. In all groups there was no indication of the activation of the ELHS in response to vagrancy. We found limited support for an increased rate of corticosterone elevation within our 3 min sample interval in a single species, but this was driven by an individual ELHS outlier. Fat scores were negatively correlated with circulating corticosterone; this relationship may suggest that ELHS activation depends upon an individual's energetic states. Interestingly, in individuals caught at the completion of an obligate long-distance flight, we found some evidence of corticosterone suppression. Although limited, data did support the induction of negative feedback mechanisms that suppress corticosterone during endurance exercise, even when fuel loads are low.

Phylogeny of the Eurasian Wren Nannus troglodytes (Aves: Passeriformes: Troglodytidae) reveals deep and complex diversification patterns of Ibero-Maghrebian and Cyrenaican populations.

The Mediterranean Basin represents a Global Biodiversity Hotspot where many organisms show high inter- and intraspecific differentiation. Extant phylogeographic patterns of terrestrial circum-Mediterranean faunas were mainly shaped through Pleistocene range shifts and range fragmentations due to retreat into different glacial refugia. Thus, several extant Mediterranean bird species have diversified by surviving glaciations in different hospitable refugia and subsequently expanded their distribution ranges during the Holocene. Such a scenario was also suggested for the Eurasian Wren (Nannus troglodytes) despite the lack of genetic data for most Mediterranean subspecies. Our phylogenetic multi-locus analysis comprised 18 out of 28 currently accepted subspecies of N. troglodytes, including all but one subspecies which are present in the Mediterranean Basin. The resulting phylogenetic reconstruction dated the onset of the entire Holarctic radiation of three Nannus species to the early Pleistocene. In the Eurasian Wren, two North African subspecies represented separate basal lineages from the Maghreb (N. t. kabylorum) and from the Libyan Cyrenaica (N. t. juniperi), being only distantly related to other Mediterranean populations. Although N. troglodytes appeared to be paraphyletic with respect to the Nearctic Winter Wren (N. hiemalis), respective nodes did not receive strong statistical support. In contrast, paraphyly of the Ibero-Maghrebian taxon N. t. kabylorum was strongly supported. Southern Iberian populations of N. t. kabylorum did not clade with Maghrebian populations of the same subspecies but formed a sister clade to a highly diverse European clade (including nominate N. t. troglodytes and eight further taxa). In accordance with a pattern also found in other birds, Eurasian populations were split into a western clade (Europe, Caucasus) and an eastern clade (Central Asia, Sino-Himalayas, East Asia). This complex phylogeographic pattern revealed cryptic diversification in N. troglodytes, especially in the Iberio-Maghrebian region.

Dense active matter model of motion patterns in confluent cell monolayers.

Epithelial cell monolayers show remarkable displacement and velocity correlations over distances of ten or more cell sizes that are reminiscent of supercooled liquids and active nematics. We show that many observed features can be described within the framework of dense active matter, and argue that persistent uncoordinated cell motility coupled to the collective elastic modes of the cell sheet is sufficient to produce swirl-like correlations. We obtain this result using both continuum active linear elasticity and a normal modes formalism, and validate analytical predictions with numerical simulations of two agent-based cell models, soft elastic particles and the self-propelled Voronoi model together with in-vitro experiments of confluent corneal epithelial cell sheets. Simulations and normal mode analysis perfectly match when tissue-level reorganisation occurs on times longer than the persistence time of cell motility. Our analytical model quantitatively matches measured velocity correlation functions over more than a decade with a single fitting parameter.

Geographic patterns of mtDNA and Z-linked sequence variation in the Common Chiffchaff and the 'chiffchaff complex'.

The Common Chiffchaff Phylloscopus collybita is an abundant, polytypic Palearctic bird. Validity of some of its subspecies is controversial and birds from some parts of the species range remain unclassified taxonomically. The relationships among populations from different geographic areas have not been sufficiently explored with molecular data. In this study we analyzed the relationships among the four species in the 'chiffchaff complex' (Common Chiffchaff, Iberian Chiffchaff P. ibericus, Canary Islands Chiffchaff P. canariensis and Mountain Chiffchaff P. sindianus), and the patterns of intraspecific geographic variation in the mtDNA ND2 gene and intron 9 of the Z-linked aconitase gene (ACO1I9) across the Common Chiffchaff range, including a recently discovered population breeding on Mt. Hermon (Anti-Lebanon mountains). Our data supported the monophyly of the chiffchaff complex and its current systematics at the species level. Within the Common Chiffchaff, the Siberian race P. c. tristis was the most differentiated subspecies and may represent a separate or incipient species. Other Common Chiffchaff subspecies also were differentiated in their mtDNA, however, lineages of neighboring subspecies formed wide zones of introgression. The Mt. Hermon population was of mixed genetic origin but contained some birds with novel unique lineage that could not be assigned to known subspecies. All Common Chiffchaff lineages diverged at the end of the Ionian stage of Pleistocene. Lineage sorting of ACO1I9 alleles was not as complete as that of mtDNA. Chiffchaff species were mostly distinct at ACO1I9, except the Common and Canary Islands Chiffchaffs that shared multiple alleles. An AMOVA identified geographic structure in Common Chiffchaff ACO1I9 variation that was broadly consistent with that of mtDNA ND2 gene. The genetic and other data suggest the chiffchaff complex to be a group of evolutionarily young taxa that represent a paradigm of 'species evolution in action' from intergrading subspecies through to apparently complete biological speciation.

The core planar cell polarity gene, Vangl2, maintains apical-basal organisation of the corneal epithelium.

The role of the core planar cell polarity (PCP) pathway protein, Vangl2, was investigated in the corneal epithelium of the mammalian eye, a paradigm anatomical model of planar cell migration. The gene was conditionally knocked out in vivo and knocked down by siRNA, followed by immunohistochemical, behavioural and morphological analysis of corneal epithelial cells. The primary defects observed in vivo were of apical-basal organisation of the corneal epithelium, with abnormal stratification throughout life, mislocalisation of the cell membrane protein, Scribble, to the basal side of cells, and partial loss of the epithelial basement membrane. Planar defects in migration after wounding and in the presence of an applied electric field were noted. However, knockdown of Vangl2 also retarded cell migration in individual cells that had no contact with their neighbours, which precluded a classic PCP mechanism. It is concluded that some of the planar polarity phenotypes in PCP mutants may arise from disruption of apical-basal polarity.

Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice.

Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU) and 5-ethynyl-2'-deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ±â€¯0.2 days) compared to 10 week old mice (4.97 ±â€¯0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ±â€¯1.4% of LESCs were shown to enter S phase in a 24 h period and were estimated to divide every 2-3 weeks. Within 24 h of corneal injury this rose significantly to 32.8 ±â€¯10.0% of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48 h period after wounding in young adults, LESC activation continued to increase (86.5 ±â€¯8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ±â€¯9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.

A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium.

OBJECTIVE: The corneas of heterozygous Pax6+/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6+/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types. RESULTS: In a preliminary study, ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/-;RCE:loxP reporter mice was also patchy. We attribute patchy Pax6 staining to mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.

Lens-regulated retinoic acid signalling controls expansion of the developing eye.

Absence of the developing lens results in severe eye defects, including substantial reductions in eye size. How the lens controls eye expansion and the underlying signalling pathways are very poorly defined. We identified RDH10, a gene crucial for retinoic acid synthesis during embryogenesis, as a key factor downregulated in the peripheral retina (presumptive ciliary body region) of lens-removed embryonic chicken eyes prior to overt reductions in eye size. This is associated with a significant decrease in retinoic acid synthesis by lens-removed eyes. Restoring retinoic acid signalling in lens-removed eyes by implanting beads soaked in retinoic acid or retinal, but not vitamin A, rescued eye size. Conversely, blocking retinoic acid synthesis decreased eye size in lens-containing eyes. Production of collagen II and collagen IX, which are major vitreal proteins, is also regulated by the lens and retinoic acid signalling. These data mechanistically link the known roles of both the lens and retinoic acid in normal eye development, and support a model whereby retinoic acid production by the peripheral retina acts downstream of the lens to support vitreous production and eye expansion.

Computer simulation of neutral drift among limbal epithelial stem cells of mosaic mice.

The use of mice that are mosaic for reporter gene expression underlies many lineage-tracing studies in stem cell biology. For example, using mosaic LacZ reporter mice, it was shown that limbal epithelial stem cells (LESCs) around the periphery of the cornea maintain radial sectors of the corneal epithelium and that radial stripe numbers declined with age. Originally, the corneal results were interpreted as progressive, age-related loss or irreversible inactivation of some LESC clones. In this study we used computer simulations to show that these results could also be explained by stochastic replacement of LESCs by neighbouring LESCs, leading to neutral drift of LESC populations. This was shown to reduce the number of coherent clones of LESCs and hence would coarsen the mosaic pattern in the corneal epithelium without reducing the absolute number of LESCs. Simulations also showed that corrected stripe numbers declined more slowly when LESCs were grouped non-randomly and that mosaicism was rarely lost unless simulated LESC numbers were unrealistically low. Possible reasons why age-related changes differ between mosaic corneal epithelia and other systems, such as adrenal cortices and intestinal crypts, are discussed.

The developmental and genetic basis of 'clubfoot' in the peroneal muscular atrophy mutant mouse.

Genetic factors underlying the human limb abnormality congenital talipes equinovarus ('clubfoot') remain incompletely understood. The spontaneous autosomal recessive mouse 'peroneal muscular atrophy' mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4-Limk1-Cfl1/cofilin-actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans.

Endogenous bioelectric currents promote differentiation of the mammalian lens.

The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and ß1 subunits of Na+ /K+ -ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (Vmem ). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized Vmem . Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of ß-crystallin, aquaporin-0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of Vmem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.

The core planar cell polarity gene, Vangl2, directs adult corneal epithelial cell alignment and migration.

This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.

Contact-mediated control of radial migration of corneal epithelial cells.

PURPOSE: Patients with a heterozygous mutation in the gene encoding the transcription factor, PAX6, have a degenerative corneal opacity associated with failure of normal radial epithelial cell migration across the corneal surface and a reported wound healing defect. This study investigated the guidance mechanisms that drive the directed migration of corneal epithelial cells. METHODS: In vivo corneal epithelial wounding was performed in adult wild-type and Pax6(+/-) mice, and the healing migration rates were compared. To investigate the control of the cell migration direction, primary corneal epithelial cells from wild-type and Pax6(+/-) mice were plated on grooved quartz substrates, and alignment relative to the grooves was assayed. A reconstructed corneal culture system was developed in which dissociated wild-type and genetically mutant corneal epithelial cells could be cultured on a de-epithelialized corneal stroma or basement membrane and their migration assayed with time-lapse microscopy. RESULTS: The Pax6(+/-) cells efficiently re-epithelialized corneal wounds in vivo but had mild slowing of healing migration compared to the wild-type. Cells aligned parallel to quartz grooves in vitro, but the Pax6(+/-) cells were less robustly oriented than the wild-type. In the reconstructed corneal culture system, corneal epithelial cells continued to migrate radially, showing that the cells are guided by contact-mediated cues from the basement membrane. Recombining wild-type and Pax6 mutant corneal epithelial cells with wild-type and Pax6 mutant corneal stroma showed that normal Pax6 dosage was required autonomously in the epithelial cells for directed migration. Integrin-mediated attachment to the substrate, and intracellular PI3Kγ activity, were required for migration. Pharmacological inhibition of cAMP signaling randomized migration tracks in reconstructed corneas. CONCLUSIONS: Striking patterns of centripetal migration of corneal epithelial cells observed in vivo are driven by contact-mediated cues operating through an intracellular cAMP pathway, and failure to read these cues underlies the migration defects that accompany corneal degeneration in patients with mutations in PAX6.

Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence.

The limbal epithelial stem cell (LESC) hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC) hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-loxP lineage tracing to distinguish between these hypotheses. Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks.

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance.

In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.

Hemizygous Le-Cre transgenic mice have severe eye abnormalities on some genetic backgrounds in the absence of LoxP sites.

Eye phenotypes were investigated in Le-Cre(Tg/-); Pax6(fl/+) mice, which were expected to show tissue-specific reduction of Pax6 in surface ectoderm derivatives. To provide a better comparison with our previous studies of Pax6(+/-) eye phenotypes, hemizygous Le-Cre(Tg/-) and heterozygous Pax6(fl/+)mice were crossed onto the CBA/Ca genetic background. After the Le-Cre transgene had been backcrossed to CBA/Ca for seven generations, significant eye abnormalities occurred in some hemizygous Le-Cre(Tg/-); Pax6(+/+) controls (without a floxed Pax6(fl) allele) as well as experimental Le-Cre(Tg/-); Pax6(fl/+) mice. However, no abnormalities were seen in Le-Cre(-/-); Pax6(fl/+) or Le-Cre(-/-); Pax6(+/+) controls (without the Le-Cre transgene). The severity and frequency of the eye abnormalities in Le-Cre(Tg/-); Pax6(+/+) control mice diminished after backcrossing Le-Cre(Tg/-) mice to the original FVB/N strain for two generations, showing that the effect was reversible. This genetic background effect suggests that the eye abnormalities are a consequence of an interaction between the Le-Cre transgene and alleles of unknown modifier genes present in certain genetic backgrounds. The abnormalities were also ameliorated by introducing additional Pax6 gene copies on a CBA/Ca background, suggesting involvement of Pax6 depletion in Le-Cre(Tg/-); Pax6(+/+) mice rather than direct action of Cre recombinase on cryptic pseudo-loxP sites. One possibility is that expression of Cre recombinase from the Pax6-Le regulatory sequences in the Le-Cre transgene depletes cofactors required for endogenous Pax6 gene expression. Our observation that eye abnormalities can occur in hemizygous Le-Cre(Tg/-); Pax6(+/+) mice, in the absence of a floxed allele, demonstrates the importance of including all the relevant genetic controls in Cre-loxP experiments.

3D MRI analysis of the lower legs of treated idiopathic congenital talipes equinovarus (clubfoot).

BACKGROUND: Idiopathic congenital talipes equinovarus (CTEV) is the commonest form of clubfoot. Its exact cause is unknown, although it is related to limb development. The aim of this study was to quantify the anatomy of the muscle, subcutaneous fat, tibia, fibula and arteries in the lower legs of teenagers and young adults with CTEV using 3D magnetic resonance imaging (MRI), and thus to investigate the anatomical differences between CTEV participants and controls. METHODOLOGY/PRINCIPAL FINDINGS: The lower legs of six CTEV (2 bilateral, 4 unilateral) and five control young adults (age 12-28) were imaged using a 3T MRI Philips scanner. 5 of the CTEV participants had undergone soft-tissue and capsular release surgery. 3D T1-weighted and 3D magnetic resonance angiography (MRA) images were acquired. Segmentation software was used for volumetric, anatomical and image analysis. Kolmogorov-Smirnov tests were performed. The volumes of the lower affected leg, muscle, tibia and fibula in unilateral CTEV participants were consistently smaller compared to their contralateral unaffected leg, this was most pronounced in muscle. The proportion of muscle in affected CTEV legs was significantly reduced compared with control and unaffected CTEV legs, whilst proportion of muscular fat increased. No spatial abnormalities in the location or branching of arteries were detected, but hypoplastic anomalies were observed. CONCLUSIONS/SIGNIFICANCE: Combining 3D MRI and MRA is effective for quantitatively characterizing CTEV anatomy. Reduction in leg muscle volume appears to be a sensitive marker. Since 5/6 CTEV cases had soft-tissue surgery, further work is required to confirm that the treatment did not affect the MRI features observed. We propose that the proportion of muscle and intra-muscular fat within the lower leg could provide a valuable addition to current clinical CTEV classification. These measures could be useful for clinical care and guiding treatment pathways, as well as treatment research and clinical audit.

Stem cells and corneal epithelial maintenance: insights from the mouse and other animal models.

Maintenance of the corneal epithelium is essential for vision and is a dynamic process incorporating constant cell production, movement and loss. Although cell-based therapies involving the transplantation of putative stem cells are well advanced for the treatment of human corneal defects, the scientific understanding of these interventions is poor. No definitive marker that discriminates stem cells that maintain the corneal epithelium from the surrounding tissue has been discovered and the identity of these elusive cells is, therefore, hotly debated. The key elements of corneal epithelial maintenance have long been recognised but it is still not known how this dynamic balance is co-ordinated during normal homeostasis to ensure the corneal epithelium is maintained at a uniform thickness. Most indirect experimental evidence supports the limbal epithelial stem cell (LESC) hypothesis, which proposes that the adult corneal epithelium is maintained by stem cells located in the limbus at the corneal periphery. However, this has been challenged recently by the corneal epithelial stem cell (CESC) hypothesis, which proposes that during normal homeostasis the mouse corneal epithelium is maintained by stem cells located throughout the basal corneal epithelium with LESCs only contributing during wound healing. In this chapter we review experimental studies, mostly based on animal work, that provide insights into how stem cells maintain the normal corneal epithelium and consider the merits of the alternative LESC and CESC hypotheses. Finally, we highlight some recent research on other stem cell systems and consider how this could influence future research directions for identifying the stem cells that maintain the corneal epithelium.

Regulation of Merkel cell development by Pax6.

Merkel cells are mechanoreceptors widely distributed in the vertebrate skin. In rodents, Merkel cells within the whisker pads are innervated by free sensory nerve endings derived from the maxillary branch of the trigeminal nerve. This study identified expression of the transcription factor Pax6 in Merkel cells and investigated its role. Immunohistochemistry and Western blot for Pax6 and Merkel cell markers, cytokeratin-8 (K8) and cytokeratin-20 (K20) were performed in wild-type and Pax6 (-/-) fetuses. The subcellular localisation of Pax6 in Merkel cells in vitro was manipulated using hydrogen peroxide. Pax6 was primarily localised within the cytoplasm of the Merkel cells at birth, but postnatally was also detected within the nuclei. In vitro, after 4 days in culture Pax6 protein was completely relocated to the nuclei of fetal-derived Merkel cells, mimicking the in vivo situation, suggesting that Pax6 acts as an active nucleo-cytoplasmic shuttling protein in common with many other homeodomain transcription factors. The subcellular localisation of Pax6 could be modulated in vitro by changing the redox potential of the culture medium for Merkel cells. Differentiation of cultured Pax6 (-/-) Merkel cells was shown to be inhibited. At perinatal stages, it was found that Pax6 is required for maintaining cytokeratin-8 expression, an early Merkel cell marker, whereas cytokeratin-20 was retained by the Pax6 (-/-) mutant cells. Pax6 is expressed in developing Merkel cells as a nucleo-cytoplasmic shuttling protein and its activity is required for normal differentiation, possibly through regulating cell maturation.

Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium.

PURPOSE: To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. METHODS: The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6).  Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway.  Reporter mice, mosaic for expression of the gene encoding ß-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo. RESULTS: Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage.  Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation.  Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh.  Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium. CONCLUSIONS: The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands.