Abstract
The mammary epithelium is indispensable for the continued survival of more than 5000 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 milk ejection. Using quantitative, multidimensional imaging of mammary cell ensembles, we reveal how stimulus-evoked Ca2+ oscillations couple to contraction in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically-deform the innermost layer of luminal cells, forcing 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 is delivered into the mouth of the dependent neonate, seconds after the command.
One Sentence Summary This study provides a window into the organization, dynamics and role of epithelial Ca2+ responses in the organ principally responsible for sustaining neonatal life in mammals.
Main Text
The ability to visualize how a single living cell, in its native environment, translates an extracellular message into an intracellular signal to execute a defined task at the cell-level and cooperatively achieve a biological outcome at the organ-level is revolutionizing our understanding of multicellular systems. Such an approach has provided new insights into a range of biological phenomena, including how plants defend against herbivory (1), how fish escape looming predators (2, 3) and how mammals store memories (4). The rational design and continued refinement of genetically-encoded Ca2+ indicators (GECIs) has fueled these advances (5). However, the use of GECIs for in situ activity mapping in vertebrates, has largely remained an achievement of neuroscience, where neural activity is tightly-coupled to intracellular Ca2+ ([Ca2+]i) signaling (6). Efforts to map activity networks in specific populations of non-excitable cells in other solid organs is lagging. Our understanding of how epithelial tissues function, for example, has principally arisen through analysis of isolated cells (often serially-propagated under physiologically-extraneous conditions), retrospective examination of fixed tissue and interrogation of genetic knockout models (where biological function is inferred in the absence of physiological redundancy or compensation).
The mammary gland has a universal and indispensable role in mammalian offspring survival. In its functionally-mature state, it consists of an inner layer of luminal (milk-producing) epithelial cells and an outer layer of contractile basal epithelial cells (7). When young offspring suckle, maternally-produced oxytocin (OT) binds to its cognate receptor (the OXTR, a Gq-linked G-protein coupled receptor) on mammary basal cells, causing them to contract (8). A model, therefore, emerges where activity may be tightly-coupled to [Ca2+]i in this organ (8–11), making functional in situ imaging of an epithelial signal-response relationship possible.
Here, we engineered mice with directed expression of a GECI to basal epithelial cells in the mammary gland. This enabled us to quantitatively probe the organization and function of real-time [Ca2+]i signaling events in individual cells within this complex living tissue, at a level of rigor that has only previously been achieved in the adult brain.
Basal cell [Ca2+]i oscillations signal to repetitively deform mammary alveoli and force milk out
We developed transgenic mice that express the fast, ultrasensitive GECI GCaMP6f (5) under the inducible control of the cytokeratin (K) 5 gene promoter (12) (GCaMP6f;K5CreERT2 mice) (Fig. 1A). The relatively high baseline fluorescence of this GECI is well-suited for the quantitative assessment of [Ca2+]i responses in functionally-mature basal cells, which are sparsely distributed with thin cellular processes (5, 13) (Fig. S1). GCaMP6f consists of a circularly permuted green fluorescent protein (GFP), enabling 3D assessment of its expression and lineage-specific localization using an anti-GFP antibody (14) and optimized methods for tissue clearing (15). Genetic recombination in this model was high (Fig. S2) and showed strong lineage-bias to basal epithelial cells (Fig. 1B).
To assess OT-mediated basal cell [Ca2+]i responses, we performed 4-dimensional (x-, y-, z-, t-) quantitative imaging of intact mammary tissue pieces from lactating GCaMP6f;K5CreERT2 mice. Tissue was loaded with the live-cell permeable dye CellTracker™ Red to visualize alveolar luminal (milk-producing) cells. A large coordinated wave of [Ca2+]i, likely due to inositol trisphosphate (InsP3)-mediated endoplasmic reticulum (ER) Ca2+ store-release (8, 9, 16), was observed in mammary basal cells following OT stimulation and its diffusion through the tissue (Fig. 1C and Movie S1). This initial transient [Ca2+]i elevation was followed by a phase of slow, stochastic [Ca2+]i oscillations (Fig. 1C and Movie S1), likely attributable to store-operated Ca2+ entry (SOCE) (9, 11).
Increases in [Ca2+]i appeared to be temporally-correlated with alveolar unit contractions. Whilst cell- and tissue-level movement is physiologically-relevant and important, it poses additional computational challenges to the analysis of single cell Ca2+ responses in 4D image sequences. To overcome this, we utilized the diffeomorphic registration approach of Advanced Normalization Tools for motion correction (17, 18). This approach corrected major tissue movements, however, alveolar unit contractions remained intact, enabling quantification of [Ca2+]i responses in thin basal cells, and analysis of the physical distortions to the alveolar units that these cells embrace. These analyses confirmed that increases in [Ca2+]i in individual basal cells were temporally correlated with physical distortions to the mechanically-compliant luminal cell layer (Fig. 1D and Fig. S3). For both the first (InsP3-mediated) response (Fig. 1E) and the subsequent oscillatory phase (Fig. 1F) increases in [Ca2+]i preceded alveolar unit contractions. No statistical difference in the firing interval for [Ca2+]i was observed between the first and second events and all subsequent events (Fig. 1G). These results reveal that each mammary alveolar unit, acting downstream of a basal cell OT-OXTR-InsP3-Ca2+ signaling axis, serves as a biological positive-displacement pump, repeatedly forcing milk out of its central lumen for passage through the ductal network.
Ca2+-contraction coupling in alveolar basal cells
To directly assess Ca2+-contraction coupling in mammary basal cells, we engineered triple transgenic mice that express GCaMP6f and the red fluorescent protein TdTomato in basal cells (GCaMP6f-TdTom;K5CreERT2 mice). Using this model, we observed increases in [Ca2+]i in single TdTomato-positive cells in response to OT, which immediately preceded their contraction (Fig. 2A-B, Fig. S4A and Movie S2). These data reveal how basal cells contract to deform the inner luminal cell layer for milk ejection and show unequivocally a temporal relationship between the Ca2+ signal and the contractile response.
The nature of alveolar basal cell contractions was also examined using 3-dimensional, deep-tissue imaging of myosin light chain (MLC) phosphorylation and activation. In OT-treated tissue, phospho-MLC (pMLC) -positive and -negative basal cells were interspersed throughout alveolar clusters (Fig. 2C), confirming the phasic and ostensibly-stochastic nature of this response. To determine whether basal contractile responses are truly random or whether there is any evidence for lobuloalveolar cooperativity in firing, we employed two agnostic approaches to analyze the functional connectivity in Ca2+ signaling events. First, we analyzed correlations in the firing pattern of individual basal cells in the post-diffusion phase and graphed the Euclidean distances between highly-correlated (> 0.5) cells. Highly-correlated responses exhibited a short Euclidean distance (Fig. 2D). Next, we analyzed network topologies by connecting highly-correlated cells within a single field-of-view. This method confirmed high clustering associated with short internodal distances in some lobular structures (small-worldness) (Fig. S4B) (19, 20). These analyses suggest some cooperativity in firing and, by extension, contraction. An important caveat of this model, however, is the constant, uniform application of OT in the bath. Physiologically, the picture is likely to be more complex, due to pulsatile bursts of OT from oxytocinergic neurons in response to infant suckling (8). Platforms to measure, in high resolution, single cell [Ca2+]i responses in conscious and actively-feeding mice, with normal maternal and social behaviors, have not yet been optimally developed.
Basal cells upregulate their contractile machinery during gestation and are licensed-to-respond to [Ca2+]i oscillations
Our quantitative image analysis revealed a two-component (signal-response) model of milk ejection, involving a GPCR-InsP3-Ca2+ signaling component linked to a contractile actin-myosin network. This prompted us to investigate developmental stage-specific variations to this model. To assess [Ca2+]i responses in mammary epithelial cells from non-pregnant mice, we created mammary organoids (21). An organoid model (retaining key structural (Fig. S5) and functional (21) features of the in vivo non-pregnant gland) was utilized in this context to overcome limitations associated with resolving single-cell [Ca2+]i responses in epithelial structures embedded deep within a light-scattering fat pad (Fig. S6). OT-stimulation of mammary organoids isolated from virgin GCaMP6f;K5CreERT2 mice produced [Ca2+]i oscillations in basal cells (Fig. 3A and Movie S3). However, [Ca2+]i oscillations were uncoupled to contraction in this model (Fig. 3B).
To investigate how this signal-response relationship may be uncoupled in the non-pregnant state, we analyzed single-cell RNA sequencing data from cells isolated from mammary glands of nulliparous, pregnant, lactating and involuting mice (22). Unsupervised clustering has previously revealed four populations of cells with a basal profile, which strongly correlates with the developmental stage of isolation (22). Comparing the “Basal-Virgin” and “Basal-Lac” clusters exposed a high number of differentially-expressed genes (Fig. 3C). The Basal-Lac cluster was significantly enriched for genes in the vascular smooth muscle contraction pathway (Fig. 3D), including myosin light chain kinase (Mylk), calponin (Cnn1) and caldesmon (Cald1) (Fig. 3E). These findings were validated by immunostaining (Fig. 3F and Fig. S7).
Both ducts and alveoli contract to expel milk in the mature gland
The lactating mouse mammary gland consists of milk-producing alveoli that are connected to the nipple via a branching ductal network. Heterogeneity in the expression of contractile markers in basal cells of ducts and alveoli has led to speculation that these two spatially-distinct cell populations are functionally-divergent (23). Immunohistochemical analysis of MYLK, CNN1 and CALD1 in small ducts, large ducts and alveoli of lactating mice (Fig. 4A) and humans (Fig. 4B) revealed that these contractile proteins are expressed at comparable levels in ducts and alveoli. We therefore examined Ca2+-contraction coupling in ductal cells of GCaMP6f-TdTom;K5CreERT2 mice at day 15.5-16.5 gestation (dpc). At this developmental stage, contractile proteins are already upregulated (Fig. 3E), Ca2+-contraction coupling is observed in alveolar structures (Movie S4) and the visualization of ducts is not encumbered by light scattering and/or absorptive properties of interposing structures. Ca2+-contraction coupling was clearly observed in ductal basal cells (Fig. 4C and Movie S5). On one occasion, a large duct from a lactating animal was able to be visualized at high cellular resolution (Movie S6), confirming these findings in the fully-mature state.
In ducts, basal cells adopt a spindle like morphology and are collectively-oriented along the length of the duct. Our data reveal that contraction of these cells generates longitudinal motion, facilitating the continued flow of milk. We also definitively demonstrate that differences in the type of motion generated by ductal and alveolar contractions stem from organizational heterogeneity—rather than divergent functional differentiation or signal transduction—resolving a longstanding unanswered question in the field.
Basal cell contractions are calcium-signal dependent with “loose” coupling
We used pharmacological tools to interrogate intracellular pathways that may be involved in Ca2+-contraction coupling in mammary basal cells. Cells from GCaMP6f-TdTom;K5CreERT2 (15.5-16.5 dpc) mice were isolated, plated in co-culture on a nanopatterned surface (Fig. 5A) and imaged within 12 h of dissection. These conditions were optimal for: 1) maintaining cell health and stage-specific differentiation, and 2) achieving anisotropy in the arrangement of contractile elements for the experimental measurement of force-generation along a single axis (24). Under these conditions, OT stimulation produced [Ca2+]i responses, which were coupled to contraction at the first (InsP3) phase (Fig. 5B-C and Movie S7). Later phase Ca2+-contraction coupling was not able to be assessed in this model, due to the intensity of the first contraction (even at pM concentrations of OT) and the relatively low strength of the newly-formed surface adhesions. Nevertheless, as Ca2+-contraction coupling is observed at this phase (Fig. 1D-E and Fig. 2A-B), the model is fit.
Intracellular Ca2+ chelation with BAPTA completely blocked [Ca2+]i responses to OT (Fig. 5C and Movie S8). Cell contractions were also blocked (Fig. 5C and Movie S8) demonstrating, unequivocally, their Ca2+-dependence. To gauge the distance between the Ca2+ source (in this case InsP3 receptors) and sensor, we compared OT-mediated basal cell contractions in cells loaded with two different [Ca2+]i chelators (BAPTA-AM and EGTA-AM), with different Ca2+ binding rates but comparable binding affinities (25, 26). Both intracellular BAPTA and EGTA were able to capture Ca2+ between the channel and the sensor (Fig 5C), suggestive of “loose” Ca2+-contraction coupling in these cells that is not strictly dependent on nanodomain signaling (26). Inhibition of the Ca2+/calmodulin-dependent MYLK (with ML-7), rho-associated protein kinase (with Y27632) or dual inhibition (ML-7 + Y27632) (27) failed to block OT-mediated basal cell contraction in this model (Fig 5C). These data suggest that, similar to vascular smooth muscle cells (28), MYLK-independent contractile pathways can also operate in functionally-mature mammary basal cells.
Distinct signaling pathways underpin the passage of milk, tears and sperm
To assess potential conservation in the signaling pathways that operate in basal cells of other OT-sensitive, fluid-transporting epithelia, we assessed OT-mediated responses in the lacrimal glands and epididymides of GCaMP6f-TdTom;K5CreERT2 mice. In the lacrimal gland, basal cells have a similar morphology, arrangement and function to mammary basal cells (29). They have previously been shown to undergo OT-dependent contractions (30), and diminished OT-OXTR signaling in these cells has been linked to dry eye disease (30). Like the mammary gland, dual expression of basal and smooth muscle markers was confirmed in lacrimal acini (Fig. 6A), however, no OT-mediated [Ca2+]i or contractile responses were detected in these cells in this study (Fig. 6B-C and Movie S9).
In males, a large burst of OT is released into the bloodstream at ejaculation (8, 31). This produces contractions of the male reproductive tract and, by assisting with the passage of fluid along this tract, these contractions are thought to reduce post-ejaculatory refractoriness and improve reproductive readiness (31, 32). Epididymal basal cells express basal cell markers, however, unlike the lacrimal and mammary glands, they do not co-express smooth muscle markers (Fig. 6D). Instead, movement of fluid through this organ appears to rely on a layer of smooth muscle surrounding the inner tubular epithelium (Fig. 6D). To assess the transport of sperm through this organ, its OT-responsiveness and its relationship to basal cell [Ca2+]i elevations, we stimulated intact epididymal tissue with a large bolus dose of OT. OT stimulation triggered marked peristaltic-like movements of the epididymal tubes (Fig. 6E) and a supra-basal pattern of phosphorylation of MLC (Fig. S8). Low frequency Ca2+ firing in basal cells was observed before and after OT-stimulation (Fig. 6F and Movie S10). Ca2+-contraction signaling can therefore be selectively uncoupled in different fluid-moving, stratified epithelia, either by variation of the signal, source or sensor.
Discussion
Real-time, in situ activity monitoring provides important insights into how individual cells behave in multi-dimensional and multi-cellular environments (1–4). This approach was used to describe and quantify the mechanism by which milk is transported through the mammary epithelium, making it available on-demand and with minimal delay to the nursing neonate (8, 33). Our data support four novel findings. Firstly, we revealed that transient [Ca2+]i elevations precede and are required for basal cell contractions in the functionally-mature mammary gland. We extended this finding to demonstrate how Ca2+-contraction coupling in a single basal cell can physically warp the layer of alveolar luminal cells that it encircles. Through this repetitive and collective (although not highly-coordinated) effort, large volumes of a thick biological emulsion can be forced through a narrow passage in a manner that is both consistent and persistent. Structure, function and expression were examined in the adjoining ductal epithelium, previously relegated to a role akin to a biological drinking straw. Instead, these analyses revealed active participation of the ductal epithelium in the process of milk ejection. Differences in the motion generated by basal cell contractions in ducts and alveoli could be ascribed to heterogeneity in cellular organization, rather than expression or function of contractile elements. Finally, we compared mammary, lacrimal and epididymal tissue in 4-dimensions, highlighting how these resembling structures utilize divergent pathways to optimally propel their biological fluid out of the mammalian body.
Funding
This work was supported by the National Health and Medical Research Council [1141008 and 1138214 (F.M.D.)], The University of Queensland (UQ FREA to F.M.D.) and the Mater Foundation (Equity Trustees / AE Hingeley Trust).
Author contributions
Conceptualization, J.W.P. and F.M.D.; Methodology, A.J.S., G.V., N.D.C., B.L.-L., E.K.S., A.D.E., T.A.S. and F.M.D.; Software, G.V., N.D.C. and A.D.E.; Formal Analysis, A.J.S., G.V., N.D.C., A.D.E. and F.M.D.; Investigation, A.J.S., T.A.S., and F.M.D.; Resources, N.M. (Komen Tissue Bank Samples), J.W.P. (mice); Writing – Original Draft, F.M.D.; Writing – Review and Editing: A.J.S., G.V., T.A.S., B.L.-L., N.M., J.W.P. and A.D.E.; Visualization, A.J.S., G.V., T.A.S., F.M.D.; Supervision, F.M.D.; Project Administration, F.M.D.; Funding Acquisition, F.M.D.
Competing interests
Authors declare no competing interests.
Data and materials availability
All data are available in the main text or the supplementary materials, or from the corresponding author upon reasonable request. The RNA sequencing data have previously been deposited in the Gene Expression Omnibus (GEO) database under accession code GSE106273. Data can also be interrogated at http://marionilab.cruk.cam.ac.uk/mammaryGland.
Acknowledgments
The authors acknowledge the Translational Research Institute (TRI) for the research space, equipment and core facilities that enabled this research. We thank the UQ Biological Resource Facility staff for help with animal work; Dr Corinne Alberthsen (Mater Research) for assistance with research ethics applications and compliance; Dr Jerome Boulanger (MRC Laboratory of Molecular Biology) for the 3D denoising algorithm; Mr Karsten Bach (University of Cambridge) for assistance with accessing and analyzing RNAseq data; and Mr Eric Pizzani (Translational Research Institute) for research computing support. Samples from the Susan G. Komen Tissue Bank at the IU Simon Cancer Center were used in this study; we thank contributors, including Indiana University (sample collection) as well as donors and their families.
Footnotes
↵† Equal contribution