Rapid mechanosensitive migration and dispersal of newly divided mesenchymal cells aid their recruitment into dermal condensates.

Embryonic mesenchymal cells are dispersed within an extracellular matrix but can coalesce to form condensates with key developmental roles. Cells within condensates undergo fate and morphological changes and induce cell fate changes in nearby epithelia to produce structures including hair follicles, feathers, or intestinal villi. Here, by imaging mouse and chicken embryonic skin, we find that mesenchymal cells undergo much of their dispersal in early interphase, in a stereotyped process of displacement driven by 3 hours of rapid and persistent migration followed by a long period of low motility. The cell division plane and the elevated migration speed and persistence of newly born mesenchymal cells are mechanosensitive, aligning with tissue tension, and are reliant on active WNT secretion. This behaviour disperses mesenchymal cells and allows daughters of recent divisions to travel long distances to enter dermal condensates, demonstrating an unanticipated effect of cell cycle subphase on core mesenchymal behaviour.

Fingerprinting of skin cells by live cell Raman spectroscopy reveals melanoma cell heterogeneity and cell-type-specific responses to UVR.

Raman spectroscopy is an emerging dermatological technique with the potential to discriminate biochemically between cell types in a label-free and non-invasive manner. Here, we use live single-cell Raman spectroscopy and principal component analysis (PCA) to fingerprint mouse melanoblasts, melanocytes, keratinocytes and melanoma cells. We show the differences in their spectra are attributable to biomarkers in the melanin biosynthesis pathway and that melanoma cells are a heterogeneous population that sit on a trajectory between undifferentiated melanoblasts and differentiated melanocytes. We demonstrate the utility of Raman spectroscopy as a highly sensitive tool to probe the melanin biosynthesis pathway and its immediate response to ultraviolet (UV) irradiation revealing previously undescribed opposing responses to UVA and UVB irradiation in melanocytes. Finally, we identify melanocyte-specific accumulation of ß-carotene correlated with a stabilisation of the UVR response in lipids and proteins consistent with a ß-carotene-mediated photoprotective mechanism. In summary, our data show that Raman spectroscopy can be used to determine the differentiation status of cells of the melanocyte lineage and describe the immediate and temporal biochemical changes associated with UV exposure which differ depending on cell type, differentiation status and competence to synthesise melanin. Our work uniquely applies Raman spectroscopy to discriminate between cell types by biological function and differentiation status while they are growing in culture. In doing so, we demonstrate for the first time its utility as a tool with which to probe the melanin biosynthesis pathway.

PLAA Mutations Cause a Lethal Infantile Epileptic Encephalopathy by Disrupting Ubiquitin-Mediated Endolysosomal Degradation of Synaptic Proteins.

During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.

Hierarchical patterning modes orchestrate hair follicle morphogenesis.

Two theories address the origin of repeating patterns, such as hair follicles, limb digits, and intestinal villi, during development. The Turing reaction-diffusion system posits that interacting diffusible signals produced by static cells first define a prepattern that then induces cell rearrangements to produce an anatomical structure. The second theory, that of mesenchymal self-organisation, proposes that mobile cells can form periodic patterns of cell aggregates directly, without reference to any prepattern. Early hair follicle development is characterised by the rapid appearance of periodic arrangements of altered gene expression in the epidermis and prominent clustering of the adjacent dermal mesenchymal cells. We assess the contributions and interplay between reaction-diffusion and mesenchymal self-organisation processes in hair follicle patterning, identifying a network of fibroblast growth factor (FGF), wingless-related integration site (WNT), and bone morphogenetic protein (BMP) signalling interactions capable of spontaneously producing a periodic pattern. Using time-lapse imaging, we find that mesenchymal cell condensation at hair follicles is locally directed by an epidermal prepattern. However, imposing this prepattern's condition of high FGF and low BMP activity across the entire skin reveals a latent dermal capacity to undergo spatially patterned self-organisation in the absence of epithelial direction. This mesenchymal self-organisation relies on restricted transforming growth factor (TGF) ß signalling, which serves to drive chemotactic mesenchymal patterning when reaction-diffusion patterning is suppressed, but, in normal conditions, facilitates cell movement to locally prepatterned sources of FGF. This work illustrates a hierarchy of periodic patterning modes operating in organogenesis.

The invasion speed of cell migration models with realistic cell cycle time distributions.

Cell proliferation is typically incorporated into stochastic mathematical models of cell migration by assuming that cell divisions occur after an exponentially distributed waiting time. Experimental observations, however, show that this assumption is often far from the real cell cycle time distribution (CCTD). Recent studies have suggested an alternative approach to modelling cell proliferation based on a multi-stage representation of the CCTD. In this paper we investigate the connection between the CCTD and the speed of the collective invasion. We first state a result for a general CCTD, which allows the computation of the invasion speed using the Laplace transform of the CCTD. We use this to deduce the range of speeds for the general case. We then focus on the more realistic case of multi-stage models, using both a stochastic agent-based model and a set of reaction-diffusion equations for the cells' average density. By studying the corresponding travelling wave solutions, we obtain an analytical expression for the speed of invasion for a general N-stage model with identical transition rates, in which case the resulting cell cycle times are Erlang distributed. We show that, for a general N-stage model, the Erlang distribution and the exponential distribution lead to the minimum and maximum invasion speed, respectively. This result allows us to determine the range of possible invasion speeds in terms of the average proliferation time for any multi-stage model.

Correction to: A Multi-stage Representation of Cell Proliferation as a Markov Process.

Equations (9) and (10) were transcribed incorrectly.

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.

A Multi-stage Representation of Cell Proliferation as a Markov Process.

The stochastic simulation algorithm commonly known as Gillespie's algorithm (originally derived for modelling well-mixed systems of chemical reactions) is now used ubiquitously in the modelling of biological processes in which stochastic effects play an important role. In well-mixed scenarios at the sub-cellular level it is often reasonable to assume that times between successive reaction/interaction events are exponentially distributed and can be appropriately modelled as a Markov process and hence simulated by the Gillespie algorithm. However, Gillespie's algorithm is routinely applied to model biological systems for which it was never intended. In particular, processes in which cell proliferation is important (e.g. embryonic development, cancer formation) should not be simulated naively using the Gillespie algorithm since the history-dependent nature of the cell cycle breaks the Markov process. The variance in experimentally measured cell cycle times is far less than in an exponential cell cycle time distribution with the same mean.Here we suggest a method of modelling the cell cycle that restores the memoryless property to the system and is therefore consistent with simulation via the Gillespie algorithm. By breaking the cell cycle into a number of independent exponentially distributed stages, we can restore the Markov property at the same time as more accurately approximating the appropriate cell cycle time distributions. The consequences of our revised mathematical model are explored analytically as far as possible. We demonstrate the importance of employing the correct cell cycle time distribution by recapitulating the results from two models incorporating cellular proliferation (one spatial and one non-spatial) and demonstrating that changing the cell cycle time distribution makes quantitative and qualitative differences to the outcome of the models. Our adaptation will allow modellers and experimentalists alike to appropriately represent cellular proliferation-vital to the accurate modelling of many biological processes-whilst still being able to take advantage of the power and efficiency of the popular Gillespie algorithm.

Abnormal corneal epithelial maintenance in mice heterozygous for the micropinna microphthalmia mutation Mp.

We investigated the corneal morphology of adult Mp/+ mice, which are heterozygous for the micropinna microphthalmia mutation, and identified several abnormalities, which implied that corneal epithelial maintenance was abnormal. The Mp/+ corneal epithelium was thin, loosely packed and contained goblet cells in older mice. Evidence also suggested that the barrier function was compromised. However, there was no major effect on corneal epithelial cell turnover and mosaic patterns of radial stripes indicated that radial cell movement was normal. Limbal blood vessels formed an abnormally wide limbal vasculature ring, K19-positive cells were distributed more widely than normal and K12 was weakly expressed in the peripheral cornea. This raises the possibilities that the limbal-corneal boundary was poorly defined or the limbus was wider than normal. BrdU label-retaining cell numbers and quantitative clonal analysis suggested that limbal epithelial stem cell numbers were not depleted and might be higher than normal. However, as corneal epithelial homeostasis was abnormal, it is possible that Mp/+ stem cell function was impaired. It has been shown recently that the Mp mutation involves a chromosome 18 inversion that disrupts the Fbn2 and Isoc1 genes and produces an abnormal, truncated fibrillin-2(MP) protein. This abnormal protein accumulates in the endoplasmic reticulum (ER) of cells that normally express Fbn2 and causes ER stress. It was also shown that Fbn2 is expressed in the corneal stroma but not the corneal epithelium, suggesting that the presence of truncated fibrillin-2(MP) protein in the corneal stroma disrupts corneal epithelial homeostasis in Mp/+ mice.

Swapping in lattice-based cell migration models.

Cell migration is frequently modeled using on-lattice agent-based models (ABMs) that employ the excluded volume interaction. However, cells are also capable of exhibiting more complex cell-cell interactions, such as adhesion, repulsion, pulling, pushing, and swapping. Although the first four of these have already been incorporated into mathematical models for cell migration, swapping has not been well studied in this context. In this paper, we develop an ABM for cell movement in which an active agent can "swap" its position with another agent in its neighborhood with a given swapping probability. We consider a two-species system for which we derive the corresponding macroscopic model and compare it with the average behavior of the ABM. We see good agreement between the ABM and the macroscopic density. We also analyze the movement of agents at an individual level in the single-species as well as two-species scenarios to quantify the effects of swapping on an agent's motility.

Genetically engineered multicistronic allele of Pmel yielding highly specific CreERT2-mediated recombination in the melanocyte lineage.

Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.

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.

Live Imaging and Analysis of Cilia and Cell Cycle Dynamics with the Arl13bCerulean-Fucci2a Biosensor and Fucci Tools.

The cell and cilia cycles are inextricably linked through the dual functions of the centrioles at both the basal body of cilia and at mitotic centrosomes. How cilia assembly and disassembly, either through slow resorption or rapid deciliation, are coordinated with cell cycle progression remains unclear in many cell types and developmental paradigms. Moreover, little is known about how additional cilia parameters including changes in ciliary length or frequency of distal tip shedding change with cell cycle stage. In order to explore these questions, we have developed the Arl13bCerulean-Fucci2a tricistronic cilia and cell cycle biosensor (Ford et al., Dev Cell 47:509-523.e7, 2018). This reporter allowed us to document the heterogeneity in ciliary behaviors during the cell cycle at a population level. Without the need for external stimuli, it revealed that in several cell types and in the developing embryo cilia persist beyond the G1/S checkpoint. Here, we describe the generation of stable cell lines expressing Arl13bCerulean-Fucci2a and open-source software to aid morphometric profiling of the primary cilium with cell cycle phases, including changes in cilium length. This resource will allow the investigation of multiple morphometric questions relating to cilia and cell cycle biology.

Melanins as Sustainable Resources for Advanced Biotechnological Applications.

Melanins are a class of biopolymers that are widespread in nature and have diverse origins, chemical compositions, and functions. Their chemical, electrical, optical, and paramagnetic properties offer opportunities for applications in materials science, particularly for medical and technical uses. This review focuses on the application of analytical techniques to study melanins in multidisciplinary contexts with a view to their use as sustainable resources for advanced biotechnological applications, and how these may facilitate the achievement of the United Nations Sustainable Development Goals.

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

BACKGROUND: The 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. RESULTS: The 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. CONCLUSION: Striping 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.

Quantitative analysis of patch patterns in mosaic tissues with ClonalTools software.

Quantitative analysis of mosaic tissues is a powerful method for following developmental lineages; however, analytical techniques are often subjective and repetitious. Here a flexible, semi-automated image analysis method for mosaic patterns is described. ClonalTools is a free customizable tool-set designed for the open-source image analysis package ImageJ. Circular, polygonal or linear one-dimensional mosaic arrays can be interrogated to provide measurements of the total number and width of positive and negative patches in a region of interest. These results are adjusted for the effects of random clumping using a previously described method to correct for differences in the contribution of the positive and negative cell type. The applicability of ClonalTools to different systems is discussed with reference to the analysis of mosaic patterns in the mouse corneal epithelium and adrenal cortex and in the outgrowth of neurites from explant cultures of mouse retina as example systems. To validate ClonalTools quantitatively, a recently published manual clonal analysis of the corneal epithelium of X-inactivation beta-Gal-mosaic mice was re-analysed. The semi-automated results did not differ significantly from the published data. Rapid quantification of such patterns to produce biologically relevant results represents a welcome improvement in terms of ease and speed of use over previous methods.

Epigenetic remodelling licences adult cholangiocytes for organoid formation and liver regeneration.

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.

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.

A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice.

The cilia and cell cycles are inextricably linked. Centrioles in the basal body of cilia nucleate the ciliary axoneme and sequester pericentriolar matrix (PCM) at the centrosome to organize the mitotic spindle. Cilia themselves respond to growth signals, prompting cilia resorption and cell cycle re-entry. We describe a fluorescent cilia and cell cycle biosensor allowing live imaging of cell cycle progression and cilia assembly and disassembly kinetics in cells and inducible mice. We define assembly and disassembly in relation to cell cycle stage with single-cell resolution and explore the intercellular heterogeneity in cilia kinetics. In all cells and tissues analyzed, we observed cilia that persist through the G1/S transition and into S/G2/M-phase. We conclude that persistence of cilia after the G1/S transition is a general property. This resource will shed light at an individual cell level on the interplay between the cilia and cell cycles in development, regeneration, and disease.