Anatomical and cellular heterogeneity in the mouse oviduct-its potential roles in reproduction and preimplantation development†.

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive organ that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are consistent coiling patterns and turning points in the coiled mouse oviduct that serve as reliable landmarks for luminal morphological regionalities. We also found previously unrecognized anatomical structures in the isthmus and uterotubal junction, which likely play roles in reproduction. Furthermore, we demarcated the ampulla-isthmus junction as a distinct region. Taken together, the oviduct mucosal epithelium has highly diverse structures with distinct epithelial cell populations, reflecting its complex functions in reproduction.

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.

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.

Acute versus chronic loss of mammalian Azi1/Cep131 results in distinct ciliary phenotypes.

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.

CD133/Prom1 marks proximal mouse oviduct epithelial progenitors and adult epithelial cells with a low generative capacity.

The epithelium lining the oviduct or fallopian tube consists of multiciliated and secretory cells, which support fertilization and preimplantation development, however, its homeostasis remains poorly understood. CD133/Prom1 expression has been used as a marker to identify adult stem cell populations in various organs and often associated with cancer cells that have stem-like properties. Using an antibody targeted to CD133 and a Cre recombinase-based lineage tracing strategy, we found that CD133/Prom1 expression is not associated with a stem/progenitor population in the oviduct but marked predominantly multiciliated cells with a low generative capacity. Additionally, we have shown that CD133 is disparately localised along the oviduct during neonatal development, and that Prom1 expressing secretory cells in the ampulla rapidly transitioned to multiciliated cells and progressively migrated to the ridge of epithelial folds.

Preparing future STEM faculty through flexible teaching professional development.

We have prepared thousands of future STEM faculty around the world to adopt evidence-based instructional practices through their participation in two massive open online courses (MOOCs) and facilitated in-person learning communities. Our novel combination of asynchronous online and coordinated, structured face-to-face learning community experiences provides flexible options for STEM graduate students and postdoctoral fellows to pursue teaching professional development. A total of 14,977 participants enrolled in seven offerings of the introductory course held 2014-2018, with 1,725 participants (11.5% of enrolled) completing the course. Our results of high levels of engagement and learning suggest that leveraging the affordances of educational technologies and the geographically clustered nature of this learner demographic in combination with online flexible learning could be a sustainable model for large scale professional development in higher education. The preparation of future STEM faculty makes an important difference in establishing high-quality instruction that meets the diverse needs of all undergraduate students, and the initiative described here can serve as a model for increasing access to such preparation.

Reprogramming Mouse Oviduct Epithelial Cells Using In Vivo Electroporation and CRISPR/Cas9-Mediated Genetic Manipulation.

Advances in gene editing tools such as CRISPR/Cas9 have made precise in vivo gene editing possible, opening up avenues of research into somatic cell reprograming to study adult stem cells, homeostasis, and malignant transformation. Here we describe a method for CRISPR/Cas9 mediated in vivo gene editing, in combination with Cre-based lineage tracing via electroporation in the mouse oviduct. This method facilitates the delivery of multiple plasmids into oviduct epithelial cells, sufficient for studying homeostasis and generation of high-grade serous ovarian cancer (HGSOC) models.

Protocol to generate mouse oviduct epithelial organoids for viral transduction and whole-mount 3D imaging.

Epithelial cells lining the oviduct/fallopian tube are essential in reproduction and have been identified as the cell-of-origin in high-grade serous ovarian carcinoma (HGSOC). This protocol describes the generation of organoids from mouse oviduct epithelial cells, providing a powerful in vitro tool to study epithelial homeostasis and malignant transformation. We also outline a protocol for whole-mount immunofluorescence and 3D confocal imaging. In addition, we describe approaches of viral transduction to investigate gene function in organoid development and epithelial cell behavior. For complete details on the use and execution of this profile, please refer to Ford et al. (2021).

Multiple cell types in the oviduct express the prolactin receptor.

Little is known about the physiological role of prolactin in the oviduct. Examining mRNA for all four isoforms of the prolactin receptor (PRLR) in mice by functional oviduct segment and stage of the estrous cycle, we found short form 3 (SF3) to be the most highly expressed, far exceeding the long form (LF) in highly ciliated areas such as the infundibulum, whereas in areas of low ciliation, the SF3 to LF ratio was ~1. SF2 expression was low throughout the oviduct, and SF1 was undetectable. Only in the infundibulum did PRLR ratios change with the estrous cycle. Immunofluorescent localization of SF3 and LF showed an epithelial (both mucosal and mesothelial) distribution aligned with the mRNA results. Despite the high SF3/LF ratio in densely ciliated regions, these regions responded to an acute elevation of prolactin (30 min, intraperitoneal), with LF-tyrosine phosphorylated STAT5 seen within cilia. Collectively, these results show ciliated cells are responsive to prolactin and suggest that prolactin regulates estrous cyclic changes in ciliated cell function in the infundibulum. Changes in gene expression in the infundibulum after prolonged prolactin treatment (7-day) showed prolactin-induced downregulation of genes necessary for cilium development/function, a result supporting localization of PRLRs on ciliated cells, and one further suggesting hyperprolactinemia would negatively impact ciliated cell function and therefore fertility. Flow cytometry, single-cell RNAseq, and analysis of LF-td-Tomato transgenic mice supported expression of PRLRs in at least a proportion of epithelial cells while also hinting at additional roles for prolactin in smooth muscle and other stromal cells.

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.

Modeling High-Grade Serous Ovarian Carcinoma Using a Combination of In Vivo Fallopian Tube Electroporation and CRISPR-Cas9-Mediated Genome Editing.

Ovarian cancer is the most lethal gynecologic cancer to date. High-grade serous ovarian carcinoma (HGSOC) accounts for most ovarian cancer cases, and it is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR-Cas9-mediated genome editing. Mutation of tumor suppressor genes associated with HGSOC in two different combinations (Brca1, Tp53, Pten with and without Lkb1) resulted in successfully generation of HGSOC, albeit with different latencies and pathophysiology. Implementing Cre lineage tracing in this system enabled visualization of peritoneal micrometastases in an immune-competent environment. In addition, these models displayed copy number alterations and phenotypes similar to human HGSOC. Because this strategy is flexible in selecting mutation combinations and targeting areas, it could prove highly useful for generating mouse models to advance the understanding and treatment of ovarian cancer. SIGNIFICANCE: This study unveils a new strategy to generate genetic mouse models of ovarian cancer with high flexibility in selecting mutation combinations and targeting areas.

Oviduct epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis.

Owing to technical advances in single-cell biology, the appreciation of cellular heterogeneity has increased, which has aided our understanding of organ function, homeostasis, and disease progression. The oviduct (also known as the fallopian tube) is the distalmost portion of the female reproductive tract. It is essential for reproduction and the proposed origin of high-grade serous ovarian carcinoma (HGSOC). In mammals, the oviduct is morphologically segmented along the ovary-uterus axis into four evolutionally conserved regions. It is unclear, however, if there is a diversification of epithelial cell characteristics between these regions. In this study, we identify transcriptionally distinct populations of secretory and multiciliated cells restricted to the distal and proximal regions of the oviduct. We demonstrate that distal and proximal populations are distinct lineages specified early in Müllerian duct development and are maintained separately. These results aid our understanding of epithelial development, homeostasis, and initiation of disease from the oviduct.

Functional Transcription Factor Target Networks Illuminate Control of Epithelial Remodelling.

Cell identity is governed by gene expression, regulated by transcription factor (TF) binding at cis-regulatory modules. Decoding the relationship between TF binding patterns and gene regulation is nontrivial, remaining a fundamental limitation in understanding cell decision-making. We developed the NetNC software to predict functionally active regulation of TF targets; demonstrated on nine datasets for the TFs Snail, Twist, and modENCODE Highly Occupied Target (HOT) regions. Snail and Twist are canonical drivers of epithelial to mesenchymal transition (EMT), a cell programme important in development, tumour progression and fibrosis. Predicted "neutral" (non-functional) TF binding always accounted for the majority (50% to 95%) of candidate target genes from statistically significant peaks and HOT regions had higher functional binding than most of the Snail and Twist datasets examined. Our results illuminated conserved gene networks that control epithelial plasticity in development and disease. We identified new gene functions and network modules including crosstalk with notch signalling and regulation of chromatin organisation, evidencing networks that reshape Waddington's epigenetic landscape during epithelial remodelling. Expression of orthologous functional TF targets discriminated breast cancer molecular subtypes and predicted novel tumour biology, with implications for precision medicine. Predicted invasion roles were validated using a tractable cell model, supporting our approach.

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.

Atomically thin layers of B-N-C-O with tunable composition.

In recent times, atomically thin alloys of boron, nitrogen, and carbon have generated significant excitement as a composition-tunable two-dimensional (2D) material that demonstrates rich physics as well as application potentials. The possibility of tunably incorporating oxygen, a group VI element, into the honeycomb sp(2)-type 2D-BNC lattice is an intriguing idea from both fundamental and applied perspectives. We present the first report on an atomically thin quaternary alloy of boron, nitrogen, carbon, and oxygen (2D-BNCO). Our experiments suggest, and density functional theory (DFT) calculations corroborate, stable configurations of a honeycomb 2D-BNCO lattice. We observe micrometer-scale 2D-BNCO domains within a graphene-rich 2D-BNC matrix, and are able to control the area coverage and relative composition of these domains by varying the oxygen content in the growth setup. Macroscopic samples comprising 2D-BNCO domains in a graphene-rich 2D-BNC matrix show graphene-like gate-modulated electronic transport with mobility exceeding 500 cm(2) V(-1) s(-1), and Arrhenius-like activated temperature dependence. Spin-polarized DFT calculations for nanoscale 2D-BNCO patches predict magnetic ground states originating from the B atoms closest to the O atoms and sizable (0.6 eV < E g < 0.8 eV) band gaps in their density of states. These results suggest that 2D-BNCO with novel electronic and magnetic properties have great potential for nanoelectronics and spintronic applications in an atomically thin platform.

S-layer anchoring and localization of an S-layer-associated protease in Caulobacter crescentus.

The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunit-subunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach. In doing so, we found that the RsaA anchoring region lies in the first approximately 225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaccharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first approximately 225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C. crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.