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Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway inDrosophila.To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss ofUbr5function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia.Ubr5loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened(Smo), a key mediator of HH signalling, dramatically enhanced theUbr5mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss ofUbr5stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations betweenUBR5expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for theUbr5gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.Author SummaryUbiquitin ligases modify proteins post-translationally which is essential for a variety of cellular processes. UBR5 is an E3 ubiquitin ligase and inDrosophilais a regulator of Hedgehog signaling. In mammals, the Hedgehog (HH) signalling pathway, among many other roles, plays an essential role in tissue maintenance, a process called homeostasis. A murine genetic system was developed to specifically eliminate UBR5 function from embryonic limb tissue that subsequently forms bone and connective tissue (ligaments and tendons). This approach revealed that UBR5 operates as a potent suppressor of excessive growth of normal cartilage and bone and prevents formation of bone in ectopic sites in connective tissue near the knees and ankle joints. In contrast to abnormal growth, UBR5 inhibits degradation of the articular cartilage that cushions the knee joint leading to extensive exposure of underlying bone. Furthermore, Ubr5 interacts with smoothened, a component of the HH pathway, identifying UBR5 as a regulator of mammalian HH signaling in the postnatal musculoskeletal system. In summary, this work shows that UBR5 interacts with the HH pathway to regulate skeletal homeostasis in and around joints of the legs and identifies targets that may be harnessed for biomedical engineering and clinical applications.

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6tm1Ued (Pax6fl) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6fl/fl and heterozygous Pax6fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6Sey-Neu (Pax6−) null allele. Pax6fl/− compound heterozygotes had more severe eye abnormalities than Pax6+/− heterozygotes, implying that Pax6fl differs from the wild-type Pax6+ allele. Immunohistochemistry showed that the Pax6fl/− corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles. Electronic supplementary material The online version of this article (doi:10.1007/s11248-016-9962-4) contains supplementary material, which is available to authorized users.

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., Graphical abstract, Highlights • We used lineage tracing to evaluate alternative corneal epithelial stem cell models. • Clones emerged from the periphery and extended centripetally as radial stripes. • This implies that active stem cells are at the periphery, not throughout the cornea. • Labelling patterns suggested stem cells cycle through quiescent periods. • More labelled clones were induced by treating younger mice.

International audience; Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using ‘real life’ in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25–26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.

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.

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.

The eye develops from three major sources of cells. The surface ectoderm of the head produces the lens and the corneal, limbal, and conjunctival epithelia. The neuroectoderm produces the neural retina and retinal pigment epithelium (RPE), and extensions of both these tissues cover the ciliary body, ciliary process, and iris. The periocular mesenchyme, comprising mesoderm and neural crest–derived mesectoderm cells, produces the choroid, sclera, corneal stroma, corneal endoderm, and stroma of the ciliary body, ciliary process, and iris.1 The transcription factor Pax6 is essential for normal eye development in vertebrates.2-6 During development Pax6 is expressed in the developing lens; conjunctival, limbal and corneal epithelia; neural retina; RPE; ciliary body; and iris.4,5 After birth, Pax6 is downregulated in many eye tissues, but expression continues in the amacrine cells of the retina and the lens and in the conjunctival, limbal, and corneal epithelia.5,7,8 Homozygous Pax6−/− mice, with two nonfunctional alleles, die at birth with no eyes or nose and with brain abnormalities.5,9-11 In some cases they have abnormal dentition but the penetrance of this effect is highly dependent on the genetic background.12 Human PAX6−/− homozygotes or compound heterozygotes are rare and the condition is lethal, causing anophthalmia with severe craniofacial and central nervous system defects.13 Heterozygous Pax6+/− mice and PAX6+/− humans produce low levels of Pax6 and are viable and fertile but have a range of eye abnormalities.2 In Pax6+/− mice, abnormal eye development commonly results in small eyes, iris hypoplasia, cataracts, a thin corneal epithelium with fewer cell layers, corneal opacity, failure of the lens to separate completely from the corneal epithelium, and glaucoma. Other developmental abnormalities, including retinal dysplasia, coloboma, abnormal cell accumulation in the vitreous, and adhesions between the lens and cornea (keratolenticular strands) or between the iris and cornea (iridocorneal synechia), may also occur.10,14-18 Adult Pax6+/− mice also show progressive corneal deterioration. The corneal epithelium is thin and fragile and goblet cells accumulate; the stroma becomes vascularized from the periphery and infiltrated with inflammatory cells.16,19 Human PAX6+/− heterozygotes usually have normal-sized eyes but otherwise show a range of abnormalities similar to those in Pax6+/− mice. Clinical conditions associated with PAX6+/− heterozygosity include aniridia (absent or hypoplastic iris), Peters’ anomaly (keratolenticular adhesions with loss of posterior cornea), iridolenticular adhesions, iridocorneal adhesions, cataract, corneal opacity, congenital nystagmus, and foveal hypoplasia.2,3,13,20,21 Postnatal changes include early-onset glaucoma, corneal vascularization, corneal infiltration by inflammatory cells (autosomal dominant keratitis), and accumulation of goblet cells in the corneal epithelium. The corneal epithelium is not maintained adequately, and this is thought to involve a deficiency of limbal stem cells.22 Eye development seems to be unusually sensitive to Pax6 dosage and both PAX6 gene duplication in humans23 and experimentally induced high expression levels in the mouse24 can also cause eye abnormalities. PAX77+/− transgenic mice are hemizygous for five to six copies of a human PAX6 yeast artificial chromosome (YAC) inserted at a single locus.24 The YAC contains the human PAX6 gene with all the enhancer and promoter elements. The amino acid sequence of human PAX6 is identical with mouse Pax6 and the human PAX6 regulatory elements are fully functional in mouse, so that the transgene is expressed in an appropriate tissue-specific manner and fully functional.24 The extra copies of PAX6 can compensate for the Pax6 deficiencies in both Pax6−/− and Pax6+/− mice. On a wild-type Pax6+/+ genetic background, however, this overexpression of Pax6 in PAX77+/− mice results in eye abnormalities, which overlap with those seen in Pax6+/− mice. These hemizygous PAX77+/− Pax6+/+ mice often had microphthalmia, small corneas; flat irides; abnormal, small or absent ciliary bodies; small abnormal lenses; and abnormal photoreceptors, but the phenotype was variable. Pax6 has multiple pleiotropic roles in cell proliferation, migration, adhesion, and signaling25 and may act cell-autonomously or nonautonomously. Experimental studies have identified several primary cell-autonomous roles for Pax6 that underlie the eye abnormalities in homozygous Pax6−/− mice.6,26-30 Some of the eye abnormalities in heterozygous Pax6+/− mice are caused by nonautonomous effects and may be secondary to primary affects of low Pax6 in the surface ectoderm and lens.14,15 Nonautonomous effects probably also underlie some of the defects caused by abnormal migration of neural crest cells18 but cell-autonomous effects may also be involved because Pax6 appears to be expressed transiently in the corneal stroma,17 and chimera experiments indicate that Pax6 acts cell-autonomously in this tissue.28 Analysis of mouse chimeras has been used successfully to distinguish between the cell-autonomous and nonautonomous effects of Pax6−/− and Pax6+/− genotypes on eye development.14,26-28 One important observation to emerge from studies of Pax6+/−↔wild-type chimeras was that Pax6+/− cells were excluded from the lens epithelium of Pax6+/−↔wild-type chimeras by embryonic day (E)16.5. Although Pax6+/− cells contributed to tissues of the anterior segment, the chimeras did not show iris hypoplasia or other anterior segment abnormalities that occur in nonchimeric Pax6+/− mice. This exclusion of Pax6+/− cells from the lens epithelium is interpreted as a cell-autonomous effect, and it produces an almost entirely wild-type lens. It was further proposed that the largely wild-type lens cells exerted a nonautonomous effect on the anterior segment and prevented the formation of anterior segment abnormalities.14 One of the purposes of this study was to determine whether PAX77+/− cells are similarly excluded from the lens. In the present study, we first examined the range of eye abnormalities caused by the PAX77+/− genotype on different genetic backgrounds. We then analyzed fetal PAX77+/−↔wild-type chimeras to investigate the underlying developmental causes of these eye abnormalities and distinguish between cell-autonomous and nonautonomous effects of elevated Pax6 expression in the eye. The use of these chimeras to demonstrate a cell-autonomous effect of increased Pax6 expression in the neurocortex has been reported elsewhere.31 Both low levels of Pax6 in Pax6+/− heterozygotes and high levels in PAX77+/− transgenics cause lens abnormalities, and so we specifically wanted to use chimeras to test the hypothesis that PAX77+/− cells would be depleted or excluded from the lens epithelium, like Pax6+/− cells in our Pax6+/−↔wild-type chimeras.

The requirement for correct dosage of the transcription factor Pax6 during corneal growth and development was investigated using the Pax6-overexpressing (PAX77) transgenic mouse. Transgenics had a microcornea phenotype due to failure of postnatal growth, associated with reduction in the number of cells layers in the corneal epithelium. Cell cycle progression was monitored using bromodeoxyuridine, p63, cyclin E, and phosphohistone-3 labeling: proliferation rates were higher in PAX77+ than wild-type, without a concomitant increase in apoptosis. Hence, failure of proliferation did not underlie microcornea. PAX77+ corneal epithelia had reduced levels of cytokeratin-12, and exhibited severe wound healing delay that, in contrast to Pax6+/- mice, could not be modulated by exogenous growth factors. PAX77+ lenses showed partial failure of lens fiber differentiation. The data demonstrate that anterior eye development is very sensitive to Pax6 dosage. Although there are similarities between the eye phenotype of Pax6 heterozygotes and overexpressing mice, there are also striking differences. Developmental

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5(LacZ/-) transgenic mice produced rare β-galactosidase (β-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the β-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6(+/-), KRT5(LacZ/-) corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6(+/-) mice.