Richard L. Mort

Lack of involvement of nucleotide excision repair gene polymorphisms in colorectal cancer

DNA repair has an essential role in protecting the genome from damage by endogenous and environmental agents. Polymorphisms in DNA repair genes and differences in repair capacity between individuals have been widely documented. For colorectal cancer, the loss of mismatch repair gene activity is a key genetic determinant. Nucleotide excision repair (NER), recombination repair (RR) and base excision repair (BER) pathways have critical roles in protection against other cancers, and we wished to investigate their role in colorectal cancer. We have compared the frequency of polymorphisms in the NER genes, XPD, XPF, XPG, ERCC1; in the BER gene, XRCC1; and in the RR gene, XRCC3; in colorectal cancer patients and in a control group. No significant associations were found for any of the NER gene polymorphisms or for the XRCC1 polymorphism. The C allele (position 18067) of the XRCC3 gene was weakly but significantly associated with colorectal cancer (odds ratio 1.52, 95% confidence interval 1.04–2.22, P=0.03). For all patients who were heterozygous for any of the repair genes studied, tumour tissue was investigated for loss of heterozygosity (LOH). Only one example of LOH was found for all the genes examined. From the association and LOH data, we conclude that these genes do not have an important role in protection against colorectal carcinogenesis.

P-Rex1 is required for efficient melanoblast migration and melanoma metastasis

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

The melanocyte lineage in development and disease

Melanocyte development provides an excellent model for studying more complex developmental processes. Melanocytes have an apparently simple aetiology, differentiating from the neural crest and migrating through the developing embryo to specific locations within the skin and hair follicles, and to other sites in the body. The study of pigmentation mutations in the mouse provided the initial key to identifying the genes and proteins involved in melanocyte development. In addition, work on chicken has provided important embryological and molecular insights, whereas studies in zebrafish have allowed live imaging as well as genetic and transgenic approaches. This cross-species approach is powerful and, as we review here, has resulted in a detailed understanding of melanocyte development and differentiation, melanocyte stem cells and the role of the melanocyte lineage in diseases such as melanoma. Summary: This Review discusses melanocyte development and differentiation, melanocyte stem cells, and the role of the melanocyte lineage in diseases such as melanoma.

Opposing Functions of the ETS Factor Family Define Shh Spatial Expression in Limb Buds and Underlie Polydactyly

Summary Sonic hedgehog (Shh) expression during limb development is crucial for specifying the identity and number of digits. The spatial pattern of Shh expression is restricted to a region called the zone of polarizing activity (ZPA), and this expression is controlled from a long distance by the cis-regulator ZRS. Here, members of two groups of ETS transcription factors are shown to act directly at the ZRS mediating a differential effect on Shh, defining its spatial expression pattern. Occupancy at multiple GABPα/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. The ETS gene family is therefore attributed with specifying the boundaries of the classical ZPA. Two point mutations within the ZRS change the profile of ETS binding and activate Shh expression at an ectopic site in the limb bud. These molecular changes define a pathogenetic mechanism that leads to preaxial polydactyly (PPD).

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

BackgroundThe mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.ResultsThe initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.ConclusionStriping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.

Activated Mutant NRasQ61K Drives Aberrant Melanocyte Signaling, Survival, and Invasiveness via a Rac1-Dependent Mechanism

Around a fifth of melanomas exhibit an activating mutation in the oncogene NRas that confers constitutive signaling to proliferation and promotes tumor initiation. NRas signals downstream of the major melanocyte tyrosine kinase receptor c-kit and activated NRas results in increased signaling via the extracellular signal–regulated kinase (ERK)/MAPK/ERK kinase/mitogen-activated protein kinase (MAPK) pathways to enhance proliferation. The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling. We tested the effects of activated NRasQ61K on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model. We find an important role for Rac1 downstream of NRasQ61K in mediating dermal melanocyte survival in vivo in mouse, but surprisingly NRasQ61K does not appear to affect melanoblast motility or proliferation during mouse embryogenesis. We also show that genetic deletion or pharmacological inhibition of Rac1 in NRasQ61K induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.

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.

Ex vivo live imaging of melanoblast migration in embryonic mouse skin

Dear Sir, Melanoblasts are the embryonic precursors of melanocytes. They are derived from the neural crest at around embryonic day 9.5 (E9.5) and upregulate early melanoblast specific markers (Mitf, Tyrosinase, Dct, Kit) around E10.5. Subsequently, melanoblasts migrate along the dorsolateral pathway throughout the developing dermis (for a recent review see Thomas and Erickson, 2008). They are distributed apparently at random throughout the epidermis at E14.5 where they begin to localise to the developing hair follicles (Mayer, 1973). Little is known about the kinetics of melanoblast migration and localisation because of the difficulty in performing confocal imaging on live embryonic skin. Culture of embryonic skin is technically challenging because of the requirement for an air-liquid-interface (ALI) in order for the tissue to develop. Historically this has been achieved by culturing on floating polycarbonate membranes (Jordan and Jackson, 2000; Kashiwagi et al., 1997). Although this method has been used successfully for bright field imaging of developing cultures (Kashiwagi et al., 1997), it is not amenable to live imaging using confocal microscopy because the samples are not sufficiently immobilised and reflection artefacts created by the tissue surface make imaging problematic (data not shown). In order to study melanoblast behaviour in the embryonic epidermis there was a requirement for: (a) a good melanoblast specific fluorescent reporter strain, (b) a method to immobilise the skin sample to prevent drift in the x, y and z dimensions, (c) a method to invert the sample so that it was compatible with a standard inverted microscope enclosure and (d) a method to maintain an ALI across the epidermal side of the tissue whilst also avoiding reflection artefacts. To achieve the first we labelled melanoblasts with yellow fluorescent protein (YFP) by combining TyrCreB mice, expressing Cre recombinase under the control of the Tyrosinase promoter (Delmas et al., 2003), with R26YFPR reporter animals that express YFP conditionally from the ROSA26 locus (Srinivas et al., 2001). Figure 1 outlines the ex vivo embryonic skin culture system we have developed to address points b to d. Briefly, we sandwiched a freshly dissected E14.5 embryonic skin sample between a Nuclepore membrane (Whatman) and a gas permeable Lumox membrane (Greiner Bio-One GmbH) so that the epidermal side of the skin was in contact with the Lumox membrane. The whole assembly is mounted in a specially designed culture chamber (epidermal side down) to allow time-lapse confocal imaging. Figure 1 An ex vivo culture system for embryonic skin. (A) An expanded schematic of the components of the culture chamber. An embryonic skin sample (E14.5) is mounted epidermal side down between an 8.0 μm Nuclepore membrane (Whatman) and a Lumox (Greiner ... In this system, the combination of the Nuclepore membrane and Matrigel provides support for the dermal side of the tissue, whilst the gas-permeable Lumox membrane allows an ALI to be maintained at the epidermal side, as well as providing a surface amenable to confocal imaging. The culture is fed from above by the diffusion of culture medium through the Matrigel and Nuclepore membrane. It should be noted that, whilst we achieved better results using a specially designed chamber to immobilise the sample, skin can be cultured in a similar configuration using 35 mm Lumox dishes (Sigma-Aldrich). In this case the skin sample is sandwiched between the base of the Lumox dish and a Nuclepore filter, ‘glued’ down using matrigel and the dish is then filled with culture medium. Figure 2(A) shows a typical field of cells from an E14.5 embryonic skin sample captured by confocal microscopy. In order to produce time-lapse series, images (single Z-planes) were captured every 7 min for up to 34 h in culture. Despite this relatively high and frequent laser exposure, skin cultures survived well and YFP expression was maintained throughout the culture period. Consequently, we were able to produce time-lapse movies of melanoblasts migrating throughout the embryonic epidermis. Video Clip S1 shows such a time-lapse experiment, in this example the culture was maintained for 8 h and images were captured every 7 min. Highly-motile melanoblasts exhibit a characteristic spindle shape and are seen to migrate apparently randomly throughout the tissue sample. Periodically cells are seen to stop migrating and round up before dividing to produce two motile daughter cells. Figure 2 Live imaging of migrating melanoblasts in embryonic skin culture. Because the skin sample is flat and at E14.5 the majority of melanoblasts are located in the epidermis a single confocal Z-section can be used to capture a field of migrating cells. (A) ... To demonstrate the importance of the ALI we disrupted it by placing an impermeable glass cover slip between the skin sample and the Lumox membrane. The resulting culture had approximately 50% of its surface area in contact with the Lumox membrane and the other 50% in contact with the cover slip. We imaged the culture so that half of the field of view was taken up by skin in contact with the Lumox membrane and the other half was skin in contact with the glass cover slip. Whilst melanoblasts cultured on a Lumox membrane remain migratory those cultured on a cover slip do not migrate and by 12-h the majority have died (see Video Clip S2). To study the kinetics of melanoblast migration we used the freeware image analysis software package ImageJ (http://rsb.info.nih.gov/ij/). The ‘Particle Tracker’ plugin is an automated Image J Plugin for multiple particle detection and tracking from digital videos that implements the algorithm described in Sbalzarini and Koumoutsakos (2005). This software allows the identification and tracking of individual cells and the plotting of their trajectories as well as providing the raw data to make calculations of speed and distance. An example is shown in Video Clip S3 and Figure 2(B, D). In summary, we describe for the first time a method for the culture of embryonic skin in a manner that allows live cell imaging of melanoblast migration. The technique enables the study of melanoblast dynamics in an ex vivo environment allowing time-lapse imaging of their interactions with one another and the developing hair follicle for the first time. Preliminary studies suggest that melanoblast migration between E14.5 and E15.5 may be random. Cells tend to switch between migrating in straight and circular trajectories and do not seem to exhibit any directional movement towards developing follicles at this age. Cells pause before undergoing cell divisions and their daughter cells appear to migrate in opposite directions after the division. Initial calculations suggest that cells migrate at speeds of around 0.5 μm/min (n = 4 trajectories), but this includes periods of minimal movement as cells pause to divide, so the actual speed may be higher. In later stages of culture (E14.5 + 34 h) cells appear to aggregate within the developing hair follicle. We anticipate that this technique will be a powerful tool in the investigation of the developmental mechanisms that control melanoblast specification, migration, survival and localisation.

Fucci2a: A bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes—there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.

Effects of Aberrant Pax6 Gene Dosage on Mouse Corneal Pathophysiology and Corneal Epithelial Homeostasis

Background Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6 +/− heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6+/Sey-Neu (Pax6+/−) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77Tg/− transgenics, which over-express Pax6 and model human PAX6 duplication. Methodology/Principal Findings We used electron microscopy to investigate ocular defects in Pax6+/− heterozygotes (low Pax6 levels) and PAX77Tg/− transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZTg/−) mice to investigate corneal epithelial maintenance by LESC clones in Pax6+/− and PAX77Tg/− mosaic mice. PAX77Tg/− mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6+/− mosaics were corrected by introducing the PAX77 transgene (in Pax6+/−, PAX77Tg/− mosaics). Pax6Leca4/+, XLacZTg/− mosaic mice (heterozygous for the Pax6Leca4 missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZTg/− mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6+/− and PAX77Tg/− mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones. Conclusions/Significance Pax6+/− and PAX77Tg/− genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.