Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

Cellular heterogeneity and dynamics of the human uterus in healthy premenopausal women.

The human uterus is a complex and dynamic organ whose lining grows, remodels, and regenerates in every menstrual cycle or upon tissue damage. Here we applied single-cell RNA sequencing to profile more the 50,000 uterine cells from both the endometrium and myometrium of 5 healthy premenopausal individuals, and jointly analyzed the data with a previously published dataset from 15 subjects. The resulting normal uterus cell atlas contains more than 167K cells representing the lymphatic endothelium, blood endothelium, stromal, ciliated epithelium, unciliated epithelium, and immune cell populations. Focused analyses within each major cell type and comparisons with subtype labels from prior studies allowed us to document supporting evidence, resolve naming conflicts, and to propose a consensus annotation system of 39 subtypes. We release their gene expression centroids, differentially expressed genes, and mRNA patterns of literature-based markers as a shared community resource. We find many subtypes show dynamic changes over different phases of the cycle and identify multiple potential progenitor cells: compartment-wide progenitors for each major cell type, transitional cells that are upstream of other subtypes, and potential cross-lineage multipotent stromal progenitors that may be capable of replenishing the epithelial, stromal, and endothelial compartments. When compared to the healthy premenopausal samples, a postpartum and a postmenopausal uterus sample revealed substantially altered tissue composition, involving the rise or fall of stromal, endothelial, and immune cells. The cell taxonomy and molecular markers we report here are expected to inform studies of both basic biology of uterine function and its disorders. SIGNIFICANCE: We present single-cell RNA sequencing data from seven individuals (five healthy pre-menopausal women, one post-menopausal woman, and one postpartum) and perform an integrated analysis of this data alongside 15 previously published scRNA-seq datasets. We identified 39 distinct cell subtypes across four major cell types in the uterus. By using RNA velocity analysis and centroid-centroid comparisons we identify multiple computationally predicted progenitor populations for each of the major cell compartments, as well as potential cross-compartment, multi-potent progenitors. While the function and interactions of these cell populations remain to be validated through future experiments, the markers and their "dual characteristics" that we describe will serve as a rich resource to the scientific community. Importantly, we address a significant challenge in the field: reconciling multiple uterine cell taxonomies being proposed. To achieve this, we focused on integrating historical and contemporary knowledge across multiple studies. By providing detailed evidence used for cell classification we lay the groundwork for establishing a stable, consensus cell atlas of the human uterus.

Cellular heterogeneity of human fallopian tubes in normal and hydrosalpinx disease states identified using scRNA-seq.

Fallopian tube (FT) homeostasis requires dynamic regulation of heterogeneous cell populations and is disrupted in infertility and ovarian cancer. Here, we applied single-cell RNA-seq to profile 59,738 FT cells from four healthy, pre-menopausal subjects. The resulting cell atlas contains 12 major cell types representing epithelial, stromal, and immune compartments. Re-clustering of epithelial cells identified four ciliated and six non-ciliated secretory epithelial subtypes, two of which represent potential progenitor pools: one leading to mature secretory cells and the other contributing to either ciliated cells or one of the stromal cell types. To understand how FT cell numbers and states change in a disease state, we analyzed 17,798 cells from two hydrosalpinx samples and observed shifts in epithelial and stromal populations and cell-type-specific changes in extracellular matrix and TGF-ß signaling; this underscores fibrosis pathophysiology. This resource is expected to facilitate future studies aimed at expanding understanding of fallopian tube homeostasis in normal development and disease.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Nonsteroidal sulfamate derivatives as new therapeutic approaches for Neurofibromatosis 2 (NF2).

BACKGROUND: Neurofibromatosis 1 and 2, although involving two different tumour suppressor genes (neurofibromin and merlin, respectively), are both cancer predisposition syndromes that disproportionately affect cells of neural crest origin. New therapeutic approaches for both NF1 and NF2 are badly needed. In promising previous work we demonstrated that two non-steroidal analogues of 2-methoxy-oestradiol (2ME2), STX3451(2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), and STX2895 (7-Ethyl-6-sulfamoyloxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline) reduced tumour cell growth and induced apoptosis in malignant and benign human Neurofibromatosis 1 (NF1) tumour cells. In earlier NF1 mechanism of action studies we found that in addition to their effects on non-classical hormone-sensitive pathways, STX agents acted on the actin- and myosin-cytoskeleton, as well as PI3Kinase and MTOR signaling pathways. Tumour growth in NF2 cells is affected by different inhibitors from those affecting NF1 growth pathways: specifically, NF2 cells are affected by merlin-downstream pathway inhibitors. Because Merlin, the affected tumour suppressor gene in NF2, is also known to be involved in stabilizing membrane-cytoskeletal complexes, as well as in cell proliferation, and apoptosis, we looked for potentially common mechanisms of action in the agents' effects on NF1 and NF2. We set out to determine whether STX agents could therefore also provide a prospective avenue for treatment of NF2. METHODS: STX3451 and STX2895 were tested in dose-dependent studies for their effects on growth parameters of malignant and benign NF2 human tumour cell lines in vitro. The mechanisms of action of STX3451 and STX2895 were also analysed. RESULTS: Although neither of the agents tested affected cell growth or apoptosis in the NF2 tumour cell lines tested through the same mechanisms by which they affect these parameters in NF1 tumour cell lines, both agents disrupted actin- and myosin-based cytoskeletal structures in NF2 cell lines, with subsequent effects on growth and cell death. CONCLUSIONS: Both STX3451 and STX2895 provide new approaches for inducing cell death and lowering tumour burden in NF2 as well as in NF1, which both have limited treatment options.

The role of jab1, a putative downstream effector of the neurotrophic cytokine macrophage migration inhibitory factor (MIF) in zebrafish inner ear hair cell development.

Macrophage migration inhibitory factor (MIF) is a neurotrophic cytokine essential for inner ear hair cell (HC) development and statoacoustic ganglion (SAG) neurite outgrowth, and SAG survival in mouse, chick and zebrafish. Another neurotrophic cytokine, Monocyte chemoattractant protein 1 (MCP1) is known to synergize with MIF; but MCP1 alone is insufficient to support mouse/chick SAG neurite outgrowth or neuronal survival. Because of the relatively short time over which the zebrafish inner ear develops (~30hpf), the living zebrafish embryo is an ideal system to examine mif and mcp1 cytokine pathways and interactions. We used a novel technique: direct delivery of antisense oligonucleotide morpholinos (MOs) into the embryonic zebrafish otocyst to discover downstream effectors of mif as well as to clarify the relationship between mif and mcp1 in inner ear development. MOs for mif, mcp1 and the presumptive mif and mcp1 effector, c-Jun activation domain-binding protein-1 (jab1), were injected and then electroporated into the zebrafish otocyst 25-48hours post fertilization (hpf). We found that although mif is important at early stages (before 30hpf) for auditory macular HC development, jab1 is more critical for vestibular macular HC development before 30hpf. After 30hpf, mcp1 becomes important for HC development in both maculae.

Chemokines and cytokines on the neuroimmunoaxis: Inner ear neurotrophic cytokines in development and disease. Prospects for repair?

The earliest stages of neuronal and sensory cell development in vertebrate sensory organs depend on "inflammatory" immune system neurotrophic cytokines/chemokines. Although classical nerve growth factors, brain-derived neurotrophic factors and glial growth factors play critical roles at various stages, the earliest directive roles belong to immune system cytokines. In frogs, fishes, birds and mammals, macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein 1 (MCP1) and RANTES, components of the otocyst-derived factor, are involved in sorting, morphogenesis, providing directional neuronal outgrowth cues as well as survival factors for both neurons and sensory cells. In this review we discuss their roles in the vertebrate inner ear.

The Vibrio cholerae var regulon encodes a metallo-ß-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor.

The genome sequence of V. cholerae O1 Biovar Eltor strain N16961 has revealed a putative antibiotic resistance (var) regulon that is predicted to encode a transcriptional activator (VarR), which is divergently transcribed relative to the putative resistance genes for both a metallo-ß-lactamase (VarG) and an antibiotic efflux-pump (VarABCDEF). We sought to test whether these genes could confer antibiotic resistance and are organised as a regulon under the control of VarR. VarG was overexpressed and purified and shown to have ß-lactamase activity against penicillins, cephalosporins and carbapenems, having the highest activity against meropenem. The expression of VarABCDEF in the Escherichia coli (ΔacrAB) strain KAM3 conferred resistance to a range of drugs, but most significant resistance was to the macrolide spiramycin. A gel-shift analysis was used to determine if VarR bound to the promoter regions of the resistance genes. Consistent with the regulation of these resistance genes, VarR binds to three distinct intergenic regions, varRG, varGA and varBC located upstream and adjacent to varG, varA and varC, respectively. VarR can act as a repressor at the varRG promoter region; whilst this repression was relieved upon addition of ß-lactams, these did not dissociate the VarR/varRG-DNA complex, indicating that the de-repression of varR by ß-lactams is indirect. Considering that the genomic arrangement of VarR-VarG is strikingly similar to that of AmpR-AmpC system, it is possible that V. cholerae has evolved a system for resistance to the newer ß-lactams that would prove more beneficial to the bacterium in light of current selective pressures.

Targeted NF1 cancer therapeutics with multiple modes of action: small molecule hormone-like agents resembling the natural anticancer metabolite, 2-methoxyoestradiol.

BACKGROUND: Both the number and size of tumours in NF1 patients increase in response to the rise in steroid hormones seen at puberty and during pregnancy. The size of tumours decreases after delivery, suggesting that hormone-targeting therapy might provide a viable new NF1 treatment approach. Our earlier studies demonstrated that human NF1 tumour cell lines either went through apoptosis or ceased growth in the presence of 2-methoxyoestradiol (2ME2), a naturally occurring anticancer metabolite of 17-ß estradiol. Previous reports of treatment with sulfamoylated steroidal and non-steroidal derivatives of 2ME2 showed promising reductions in tumour burden in hormone-responsive cancers other than NF1. Here we present the first studies indicating that 2ME2 derivatives could also provide an avenue for treating NF1, for which few treatment options are available. METHODS: STX3451, (2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), a non-steroidal sulphamate analogue of 2ME2, was tested in dose-dependent studies of malignant and benign NF1 human tumour cell lines and cell lines with variable controlled neurofibromin expression. The mechanisms of action of STX3451 were also analysed. RESULTS: We found that STX3451-induced apoptosis in human malignant peripheral nerve sheath tumour (MPNST) cell lines, even in the presence of elevated oestrogen and progesterone. It inhibits both PI3 kinase and mTOR signalling pathways. It disrupts actin- and microtubule-based cytoskeletal structures in cell lines derived from human MPNSTs and in cells derived from benign plexiform neurofibromas. STX3451 selectively kills MPNST-derived cells, but also halts growth of other tumour-derived NF1 cell lines. CONCLUSION: STX3451 provides a new approach for inducing cell death and lowering tumour burden in NF1 and other hormone-responsive cancers with limited treatment options.

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear.

This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.

The cytokine macrophage migration inhibitory factor (MIF) acts as a neurotrophin in the developing inner ear of the zebrafish, Danio rerio.

Macrophage migration inhibitory factor (MIF) plays versatile roles in the immune system. MIF is also widely expressed during embryonic development, particularly in the nervous system, although its roles in neural development are only beginning to be understood. Evidence from frogs, mice and zebrafish suggests that MIF has a major role as a neurotrophin in the early development of sensory systems, including the auditory system. Here we show that the zebrafish mif pathway is required for both sensory hair cell (HC) and sensory neuronal cell survival in the ear, for HC differentiation, semicircular canal formation, statoacoustic ganglion (SAG) development, and lateral line HC differentiation. This is consistent with our findings that MIF is expressed in the developing mammalian and avian auditory systems and promotes mouse and chick SAG neurite outgrowth and neuronal survival, demonstrating key instructional roles for MIF in vertebrate otic development.

Direct delivery of MIF morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development.

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A role for juvenile hormone in the prepupal development of Drosophila melanogaster.

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met(27) larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis.

A student team in a University of Michigan biomedical engineering design course constructs a microfluidic bioreactor for studies of zebrafish development.

The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008).

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio).

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.

Cadherin-4 plays a role in the development of zebrafish cranial ganglia and lateral line system.

We previously reported that cadherin-4 (also called R-cadherin) was expressed by the majority of the developing zebrafish cranial and lateral line ganglia. Cadherin-4 (Cdh4) function in the formation of these structures in zebrafish was studied using morpholino antisense technology. Differentiation of the cranial and lateral line ganglia and lateral line nerve and neuromasts of the cdh4 morphants was analyzed using multiple neural markers. We found that a subset of the morphant cranial and lateral line ganglia were disorganized, smaller, with reduced staining, and/or with altered shape compared to control embryos. Increased cell death in the morphant ganglia likely contributed to these defects. Moreover, cdh4 morphants had shorter lateral line nerves and a reduced number of neuromasts, which was likely caused by disrupted migration of the lateral line primordia. These results indicate that Cdh4 plays a role in the normal formation of the zebrafish lateral line system and a subset of the cranial ganglia.

Cadherin-2 participates in the morphogenesis of the zebrafish inner ear.

Molecular mechanisms that control inner ear morphogenesis from the placode to the three-dimensional functional organ are not well understood. We hypothesize that cell-cell adhesion, mediated by cadherin molecules, contributes significantly to various stages of inner ear formation. Cadherin-2 (Cdh2) function during otic vesicle morphogenesis was investigated by examining morpholino antisense oligonucleotide knockdown and glass onion (glo) (Cdh2 mutant) zebrafish embryos. Placode formation, vesicle cavitation and specification occurred normally, but morphogenesis of the otic vesicle was affected by Cdh2 deficiency: semicircular canals were reduced or absent. Phalloidin staining of the hair cell stereocillia demonstrated that cadherin-2 (cdh2) loss-of-function did not affect hair cell number, but acetylated tubulin labeling showed that hair cell kinocilia were shorter and irregularly shaped. Statoacoustic ganglion size was significantly reduced, which suggested that neuron differentiation or maturation was affected. Furthermore, cdh2 loss-of-function did not cause a general developmental delay, since differentiation of other tissues, including eye, proceeded normally. These findings demonstrate that Cdh2 selectively affects epithelial morphogenetic cell movements, particularly semicircular canal formation, during normal ear mophogenesis.

Zebrafish cone-rod (crx) homeobox gene promotes retinogenesis.

The mammalian Cone-rod homeobox (Crx) gene is a divergent member of the Otx gene family known to be involved in differentiation and survival of retinal photoreceptors and photoentrainment of circadian rhythms. Zebrafish have two genes in the Otx5/crx orthology class, and we previously showed that crx can transactivate rhodopsin expression in vitro, and that otx5 (orthodenticle-related gene), but not crx, regulates expression of circadian genes in the pineal. Here, we show that zebrafish crx does not regulate expression of opsins and other photoreceptor-specific genes in the pineal. We further show that crx is expressed in proliferating retinal progenitors and may be involved in patterning the early optic primordium and in promoting the differentiation of retinal progenitors, including photoreceptors. These results suggest novel functions for zebrafish crx during retinal specification and differentiation.

Otx5 regulates genes that show circadian expression in the zebrafish pineal complex.

The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus-the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. Our results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex.