Optopharmacological tools for precise spatiotemporal control of oxytocin signaling in the central nervous system and periphery.

Oxytocin is a neuropeptide critical for maternal physiology and social behavior, and is thought to be dysregulated in several neuropsychiatric disorders. Despite the biological and neurocognitive importance of oxytocin signaling, methods are lacking to activate oxytocin receptors with high spatiotemporal precision in the brain and peripheral mammalian tissues. Here we developed and validated caged analogs of oxytocin which are functionally inert until cage release is triggered by ultraviolet light. We examined how focal versus global oxytocin application affected oxytocin-driven Ca2+ wave propagation in mouse mammary tissue. We also validated the application of caged oxytocin in the hippocampus and auditory cortex with electrophysiological recordings in vitro, and demonstrated that oxytocin uncaging can accelerate the onset of mouse maternal behavior in vivo. Together, these results demonstrate that optopharmacological control of caged peptides is a robust tool with spatiotemporal precision for modulating neuropeptide signaling throughout the brain and body.

Combined Inhibition of G9a and EZH2 Suppresses Tumor Growth via Synergistic Induction of IL24-Mediated Apoptosis.

G9a and EZH2 are two histone methyltransferases commonly upregulated in several cancer types, yet the precise roles that these enzymes play cooperatively in cancer is unclear. We demonstrate here that frequent concurrent upregulation of both G9a and EZH2 occurs in several human tumors. These methyltransferases cooperatively repressed molecular pathways responsible for tumor cell death. In genetically distinct tumor subtypes, concomitant inhibition of G9a and EZH2 potently induced tumor cell death, highlighting the existence of tumor cell survival dependency at the epigenetic level. G9a and EZH2 synergistically repressed expression of genes involved in the induction of endoplasmic reticulum (ER) stress and the production of reactive oxygen species. IL24 was essential for the induction of tumor cell death and was identified as a common target of G9a and EZH2. Loss of function of G9a and EZH2 activated the IL24-ER stress axis and increased apoptosis in cancer cells while not affecting normal cells. These results indicate that G9a and EZH2 promotes the evasion of ER stress-mediated apoptosis by repressing IL24 transcription, therefore suggesting that their inhibition may represent a potential therapeutic strategy for solid cancers. SIGNIFICANCE: These findings demonstrate a novel role for G9a and EZH2 histone methyltransferases in suppressing apoptosis, which can be targeted with small molecule inhibitors as a potential approach to improve solid cancer treatment.

Mammary basal cells: Stars of the show.

Nearly all mammals rely on lactation to support their young and to ensure the continued survival of their species. Despite its importance, relatively little is known about how milk is produced and how it is ejected from the lumen of mammary alveoli and ducts. This review focuses on the latter. We discuss how a relatively small number of basal cells, wrapping around each alveolar unit, contract to forcibly expel milk from the alveolar lumen. We consider how individual basal cells coordinate their activity, the fate of these cells at the end of lactation and avenues for future deliberation and exploration.

CSF1R-dependent macrophages control postnatal somatic growth and organ maturation.

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.

The first junior European Calcium Society meeting: calcium research across scales, Kingdoms and countries.

The first junior European Calcium Society online meeting, held October 20-21, 2020, aimed to promote junior researchers in the Ca2+ community. The meeting included four scientific sessions, covering Ca2+ research from molecular detail to whole organisms. Each session featured one invited speaker and three speakers selected based on submitted abstracts, with the overall aim of actively involving early-career researchers. Consequently, the meeting underlined the diversity of Ca2+ physiology, by showcasing research across scales and Kingdoms, as presented by a correspondingly diverse speaker panel across career stages and countries. In this meeting report, we introduce the visions of the junior European Calcium Society board and summarize the meeting content.

Mammary mechanobiology - investigating roles for mechanically activated ion channels in lactation and involution.

The ability of a mother to produce a nutritionally complete neonatal food source has provided a powerful evolutionary advantage to mammals. Milk production by mammary epithelial cells is adaptive, its release is exquisitely timed, and its own glandular stagnation with the permanent cessation of suckling triggers the cell death and tissue remodeling that enables female mammals to nurse successive progeny. Chemical and mechanical signals both play a role in this process. However, despite this duality of input, much remains unknown about the nature and function of mechanical forces in this organ. Here, we characterize the force landscape in the functionally mature gland and the capacity of luminal and basal cells to experience and exert force. We explore molecular instruments for force-sensing, in particular channel-mediated mechanotransduction, revealing increased expression of Piezo1 in mammary tissue in lactation and confirming functional expression in luminal cells. We also reveal, however, that lactation and involution proceed normally in mice with luminal-specific Piezo1 deletion. These findings support a multifaceted system of chemical and mechanical sensing in the mammary gland, and a protective redundancy that ensures continued lactational competence and offspring survival.

Got Milk? Identifying and Characterizing Lactation Defects in Genetically-Engineered Mouse Models.

The ability to produce and expel milk is important for the health and survival of all mammals. Nevertheless, our understanding of the molecular events underlying the execution of this process remains incomplete. Whilst impaired mammary gland development and lactational competence remains the subject of focused investigations, defects in these events may also be an unintended consequence of genetic manipulation in rodent models. In this technical report, we outline established and emerging methods to characterize lactation phenotypes in genetically-engineered mouse models. We discuss important considerations of common models, optimized conditions for mating and the importance of litter size and standardization. Methods for quantifying milk production and quality, as well as protocols for wholemount preparation, immunohistochemistry and the preparation of RNA and protein lysates are provided. This review is intended to help guide researchers new to the field of mammary gland biology in the systematic analysis of lactation defects and in the preparation of samples for more focused mechanistic investigations.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

A Primary Cell and Organoid Platform for Evaluating Pharmacological Responses in Mammary Epithelial Cells.

An essential process in predicting the in vivo pharmacological activity of a candidate molecule involves the evaluation of target responses using established model systems. While these models largely comprise immortalized cells, which are often serially passaged as monolayers on uniformly stiff substrates and are modified to overexpress one or more components of the pathway-of-interest, the importance of cell identity, heterogeneity, and three-dimensional (3D) context to target response is gaining increasing attention. Here, we assess intracellular calcium responses in mouse mammary epithelial cells in three distinct model systems: 3D primary organoids, 2D primary epithelial cells, and 2D immortalized cells. Specifically, we assess intracellular calcium responses to a number of extracellular signals implicated in the regulation of basal (or myoepithelial) cell function. These findings provide further insights into cell type and context-specific pharmacological responses in mammary epithelial cells and highlight the opportunities and challenges in the adoption of architecturally complex and heterogeneous in vitro assays in pharmacological research.

Developmental Stage-Specific Distribution of Macrophages in Mouse Mammary Gland.

Mammary gland development begins in the embryo and continues throughout the reproductive life of female mammals. Tissue macrophages (Mϕs), dependent on signals from the Mϕ colony stimulating factor 1 receptor (CSF1R), have been shown to regulate the generation, regression and regeneration of this organ, which is central for mammalian offspring survival. However, the distribution of Mϕs in the pre- and post-natal mammary gland, as it undergoes distinct phases of development and regression, is unknown or has been inferred from immunostaining of thin tissue sections. Here, we used optical tissue clearing and 3-dimensional imaging of mammary tissue obtained from Csf1r-EGFP mice. Whilst tissue Mϕs were observed at all developmental phases, their abundance, morphology, localization and association with luminal and basal epithelial cells exhibited stage-specific differences. Furthermore, sexual dimorphism was observed at E14.5, when the male mammary bud is severed from the overlying epidermis. These findings provide new insights into the localization and possible functions of heterogeneous tissue Mϕ populations in mammogenesis.

Formation and Function of Mammalian Epithelia: Roles for Mechanosensitive PIEZO1 Ion Channels.

Mechanical forces play important roles in shaping mammalian development. In the embryo, cells experience force both during the formation of the mammalian body plan and in the ensuing phase of organogenesis. Physical forces - including fluid flow, compression, radial pressure, contraction, and osmotic pressure - continue to play central roles as organs mature, function, and ultimately dysfunction. Multiple mechanisms exist to receive, transduce, and transmit mechanical forces in mammalian epithelial tissues and to integrate these cues, which can both fluctuate and coincide, with local and systemic chemical signals. Drawing near a decade since the discovery of the bona fide mechanically activated ion channel, PIEZO1, we discuss in this mini-review established and emerging roles for this protein in the form and function of mammalian epithelia.

An element for development: Calcium signaling in mammalian reproduction and development.

Life begins with calcium. It is the language that a sperm cell uses to respond to instructions from the female reproductive tract to alter its swimming pattern and gain the force required to penetrate the outer layers of the oocyte. The first heartbeat transpires from spontaneous calcium oscillations in embryonic cardiomyocytes. The dynamic balance of calcium between auditory hair cells and the fluid they bathe in enables us to hear our first sound, and our interpretation and response to this sound requires rapid calcium flux through neuronal voltage-sensitive calcium channels. Calcium signaling can decode and integrate informational cues from both the chemical and mechanical cellular microenvironment to drive the form and function of many mammalian organ-systems. Here, we highlight roles for the intracellular calcium signal in the reproductive- and developmental- biology of mammals. A greater appreciation of the signaling pathways that initiate and support life has wide-ranging significance for the fields of reproductive science, neonatology and regenerative medicine. Furthermore, as developmental programs are often reactivated in cancer, an improved understanding of the signaling pathways that underpin mammalian development has important implications for cancer research.

Development of a high-throughput fluorescent no-wash sodium influx assay.

Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 µM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 µM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

Neutral lineage tracing of proliferative embryonic and adult mammary stem/progenitor cells.

Mammary gland development occurs over multiple phases, beginning in the mammalian embryo and continuing throughout reproductive life. The remarkable morphogenetic capacity of the mammary gland at each stage of development is attributed to the activities of distinct populations of mammary stem cells (MaSCs) and progenitor cells. However, the relationship between embryonic and adult MaSCs, and their fate during different waves of mammary gland morphogenesis, remains unclear. By employing a neutral, low-density genetic labelling strategy, we characterised the contribution of proliferative stem/progenitor cells to embryonic, pubertal and reproductive mammary gland development. Our findings further support a model of lineage restriction of MaSCs in the postnatal mammary gland, and highlight extensive redundancy and heterogeneity within the adult stem/progenitor cell pool. Furthermore, our data suggest extensive multiplicity in their foetal precursors that give rise to the primordial mammary epithelium before birth. In addition, using a single-cell labelling approach, we revealed the extraordinary capacity of a single embryonic MaSC to contribute to postnatal ductal development. Together, these findings provide tantalising new insights into the disparate and stage-specific contribution of distinct stem/progenitor cells to mammary gland development.

Mammary Stem Cells: Premise, Properties, and Perspectives.

Adult mammary stem cells (MaSCs) drive postnatal organogenesis and remodeling in the mammary gland, and their longevity and potential have important implications for breast cancer. However, despite intense investigation the identity, location, and differentiation potential of MaSCs remain subject to deliberation. The application of genetic lineage-tracing models, combined with quantitative 3D imaging and biophysical methods, has provided new insights into the mammary epithelial hierarchy that challenge classical definitions of MaSC potency and behaviors. We review here recent advances - discussing fundamental unresolved properties of MaSC potency, dynamics, and plasticity - and point to evolving technologies that promise to shed new light on this intractable debate. Elucidation of the physiological mammary differentiation hierarchy is paramount to understanding the complex heterogeneous breast cancer landscape.

The ins and outs of calcium signalling in lactation and involution: Implications for breast cancer treatment.

The mammary epithelium is highly responsive to hormonal and non-hormonal signalling cues for physiological growth, function and tissue remodelling. Whilst steroid hormones freely diffuse across the cell membrane to bind to intracellular hormone receptors, cell-impermeable ligands, including many peptide hormones, growth factors and cytokines, bind to receptors on the plasma membrane and relay their message via the specific activation of intracellular signal transduction pathways. A signalling pathway that is indispensable for decoding many extracellular signals into cellular responses is calcium (Ca2+). Changes in the expression of specific Ca2+ channels, pumps and binding proteins may therefore greatly alter the nature of the cellular response to various growth, morphogenetic and cell death stimuli. This review summarises changes in the expression, localisation and function of key Ca2+ channels and pumps in mammary epithelial cells during lactation and discusses how this altered Ca2+ handling may later expose these cells to targeted cell death during post-lactational involution. A greater understanding of the processes regulating the growth, death and regeneration of the mammary epithelium under physiological conditions may provide important insights into the proliferation and survival mechanisms underpinning malignant growth. The therapeutic manipulation of specific calcium signalling pathways in breast cancer cells to control aberrant cell proliferation and/or turnover represents an aim for the future.