Significance Many questions about how stem cells communicate with neighboring cells and self-organize to initiate tissue formation remain unanswered. We uncovered mechanisms employed by embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) to coform embryo-like structures. We describe ESC-generated cytonemes that react to self-renewal–promoting Wnt ligands secreted by TSCs. We identified glutamatergic activity upon formation of ESC–TSC interaction. This cellular connection is required for the transmission of Wnt signals to ESCs for Wnt/β-catenin pathway activation, a process that regulates morphogenesis. Given that many stem cell types express glutamate receptors and rely on niche-secreted Wnt ligands for self-renewal, we propose that Wnt and glutamatergic signaling crosstalk may prove prevalent in various mammalian tissues to regulate stem cell–niche interactions.

Abstract Spatial cellular organization is fundamental for embryogenesis. Remarkably, coculturing embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) recapitulates this process, forming embryo-like structures. However, mechanisms driving ESC–TSC interaction remain elusive. We describe specialized ESC-generated cytonemes that react to TSC-secreted Wnts. Cytoneme formation and length are controlled by actin, intracellular calcium stores, and components of the Wnt pathway. ESC cytonemes select self-renewal–promoting Wnts via crosstalk between Wnt receptors, activation of ionotropic glutamate receptors (iGluRs), and localized calcium transients. This crosstalk orchestrates Wnt signaling, ESC polarization, ESC–TSC pairing, and consequently synthetic embryogenesis. Our results uncover ESC–TSC contact–mediated signaling, reminiscent of the glutamatergic neuronal synapse, inducing spatial self-organization and embryonic cell specification.

Stem cells reside in cellular niches that impart chemical and physical signals to regulate their self-renewal and differentiation (1). Understanding the communication between the niche and stem cells paved the way to engineering organoids that mimic in vivo tissue structures. Organoids are invaluable to the study of development and tissue patterning, for drug screening, and for regenerative medicine applications. Synthetic embryos that resemble the blastocyst (2) and the gastrulating embryo (3, 4) have recently been described. These structures offer unique insights into processes of morphogenesis and patterning that can be challenging to study in the naturally developing embryo. To generate synthetic embryos, embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) are mixed and allowed to self-sort and organize to develop the embryonic structure.

Wnt/β-catenin signaling has been implicated in tissue patterning and the self-renewal of many types of mammalian stem cells, including embryonic stem cells (5). This pathway is regulated by Wnt ligands, which are often secreted locally from the stem cell niche (6). Wnt proteins bind to the low-density lipoprotein-related receptors 5 and 6 (LRP5/6) and to a member of the Frizzled (Fzd) receptor family. Ligand binding induces phosphorylation of the cytoplasmic tail of LRP6, the binding of Disheveled protein (DVL) to the cytoplasmic domain of Fzd, and the inhibition of the destruction complex that targets β-catenin for degradation. Consequently, β-catenin is stabilized, translocates to the nucleus, and initiates the Wnt-mediated transcription program. In the context of many mammalian stem cells, this program blocks differentiation and promotes self-renewal (7).

The secretion of Wnt ligands must be spatially and temporally controlled to provide local cues that regulate the abundance of tissue stem cells and the differentiation of progeny cells once exiting the niche (8). To produce position-dependent information during a specific time frame, diffusible morphogenetic gradients are a seemingly implausible mechanism because they lack precision and temporal dynamics (9). Conversely, contact-dependent signaling ensures the transfer of the developmental signal via direct cell–cell contact (10). In 1999, Ramírez-Weber and Kornberg identified cytonemes as signaling filopodia that orient toward morphogen-producing cells and specialize in recruiting developmental signals (11). Others have also identified cytonemes made by ligand-producing cells, which extend the ligand toward responsive cells (12⇓–14). Both cytoneme types limit ligand dispersion and effectively facilitate ligand delivery to the responding cells. Cytonemes exist in Drosophila tissues and in other organisms, including Zebrafish (15), vertebrate embryos (16), and cultured human cells (17).

Mechanisms that regulate ESC–TSC communication and their spatial organization to generate synthetic embryos are incompletely defined. Additionally, knowledge of how mammalian stem cells distinguish and receive niche signals to facilitate their division and determine cell fate remains elusive. To address these issues, we followed the interaction between ESCs and TSCs at single-cell resolution. We found that ESCs extend cytonemes that can contact TSCs and recognize secreted Wnts, resulting in ESC–TSC pairing. When Wnt ligand secretion in TSCs was inhibited, ESC–TSC pairing and consequently the formation of synthetic embryos significantly decreased.

We investigated whether the cytonemes of ESCs distinguish between Wnt ligands that activate the Wnt/β-catenin pathway (e.g., Wnt3a) versus other Wnts that transduce β-catenin–independent pathways (e.g., Wnt5a). Therefore, we immobilized purified Wnt3a and Wnt5a onto microbeads, distributed the microbeads around single ESCs, and investigated the interaction between cytonemes and Wnt beads. Our results indicate that ESCs can distinguish between signals and selectively reinforce a connection to the self-renewal Wnt3a ligand in an LRP6-dependent process. This signal recruitment is also mediated by the activity of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors at the cytonemes, which produces calcium transients. We identified the roles of intracellular calcium stores, Wnt receptors, DVL2, and β-catenin in regulating the formation and length of ESC cytonemes.

In conclusion, we demonstrate that ESCs possess specialized cytonemes that react to self-renewal signals and orchestrate ESC–TSC pairing, setting the basis for spatial organization and specification of embryonic tissues.

ESCs Extend Cytonemes to Initiate Contact with TSCs ESCs and TSCs possess the ability to self-sort and organize when cultured together to generate embryonic structures (2⇓–4). By time-lapse imaging, we investigated how the initial interaction between cell types was achieved. Single TSCs, which constitutively expressed enhanced green fluorescent protein (eGFP), displayed limited movement (Fig. 1A). We used ESCs expressing the F-actin reporter Ftractin-mRuby (18), permitting visualization of fine membranous structures during ESC–TSC interactions. We observed single ESCs extending protrusions that transiently contacted TSCs. After the initial contact, ESCs reacted by directing a larger protrusion to establish a stable contact with TSCs (reactive interaction; RI), often followed by ESC–TSC pairing (Fig. 1A and Movie S1). We did not observe TSCs contacting ESCs in a similar manner to establish ESC–TSC pairing. Fig. 1. ESCs selectively react to self-renewal–promoting Wnt signals and initiate pairing with TSCs. (A) Representative frames from time-lapse imaging of ESCs (expressing Ftractin-mRuby3; magenta) interacting with TSCs that express eGFP (green). Examples of reactive interactions (Left, green) and nonreactive interactions (Right, red) are shown. (Scale bars, 20 μm.) Time is expressed in minutes. Arrowheads (yellow) indicate initial interaction through thin protrusions; the arrow (white) indicates larger protrusion. (B) Quantification of the percentage of reactive (green) and nonreactive (red) interactions between ESCs and TSCs in different conditions. n ≥ 44 from more than three independent experiments. (C) Schematic (Left) and representative images (Right) of ESCs expressing the 7xTCF-EGFP//simian virus 40 early promoter (SV40)-mCherry (a Wnt/β-catenin pathway reporter) and contacting TSCs. Shown are representative images of EGFP expression levels in ESCs contacting WT cells (CNTRL) or 24-h IWP2-pretreated TSCs. In both conditions, 100 ng/mL R-spondin was added to the media to increase pathway activation. Dashed white line marks TSCs (Top) and ESCs (Bottom). (Scale bars, 20 µm.) Time is expressed in hours. (D) Representative images from time-lapse imaging of ESCs expressing the F-actin reporter Ftractin-mRuby3 (Bottom, grayscale) and interacting with beads. Reactive (Left, green) and nonreactive (Right, red) interactions are shown. (Scale bars, 20 μm.) Time is expressed in minutes. Insets are magnified and contrast-enhanced for clarity. (E) Quantification of the percentage of reactive (green) and nonreactive (red) interactions between ESCs and different types of beads. n ≥ 41 cells from at least three independent experiments. Asterisks indicate statistical significance calculated by Fisher’s exact test: ***P < 0.001; ****P < 0.0001. ESCs rely on activation of the Wnt/β-catenin pathway for self-renewal (19, 20). Therefore, we investigated whether TSCs secrete Wnt ligands that are received by ESCs. We profiled the transcripts of the 19 Wnt genes in TSCs, showing the expression of 16 Wnt transcripts (SI Appendix, Fig. S1A). Importantly, the interaction of ESCs and TSCs can result in activation of the Wnt/β-catenin pathway in ESCs, as indicated by the 7 oligomerized T cell factor (TCF)-binding sites (7xTCF)-eGFP (21) ESC reporter line (Fig. 1C and SI Appendix, Fig. S1B). To verify if the TSCs were, in fact, the source of the Wnt ligands, we opted to use a short-term inhibition of Wnt ligand secretion to minimize the potential impact on TSC maintenance and identity. Accordingly, ESCs incubated with TSCs pretreated with inhibitor of Wnt production-2 (IWP2), a small molecule that blocks the secretion of Wnt ligands (22), for 24 h significantly reduced the magnitude of activation, similar to that of ESCs cultured alone (Fig. 1C and SI Appendix, Fig. S1B). These results indicate that TSCs produce Wnt ligands that are received by ESCs to activate the Wnt/β-catenin pathway. Next, we determined whether the ESC–TSC interaction itself is affected by IWP2 treatment of TSCs. We observed that ESCs contact treated TSCs transiently via the protrusions; however, in 76% of cases this was not followed by ESC–TSC pairing (nonreactive interaction; Fig. 1 A and B and Movie S2). We obtained similar results using a different Wnt secretion inhibitor, Wnt-C59 (ref. 23, Fig. S1C). We speculated that the ESC protrusions are cytonemes that sense TSC-derived Wnt ligands, which are essential for the establishment of stable contacts during ESC–TSC pairing. To confirm this, we generated a double knock-out (dKO) of the Wnt coreceptors LRP5 and LRP6 in ESCs (LRP5/6dKO) and observed that the transient contact between cytonemes and TSCs was unaffected. However, these ESCs had a significantly reduced ability to establish stable contacts with TSCs, similarly to the ESC interaction with IWP2-pretreated TSCs (Fig. 1B). Furthermore, both IWP2 (or Wnt-C59) treatment and LRP5/6dKO ESCs resulted in a significant reduction in the formation of synthetic embryo structures (3) in three-dimensional (3D) culture (SI Appendix, Fig. S2). Our results suggest that specialized ESC cytonemes induce ESC–TSC pairing, an essential step in synthetic embryogenesis. To study the specificity of these cytonemes for Wnt ligands, we covalently immobilized purified Wnts to microbeads and investigated the cytoneme-bead interactions.

ESCs Selectively Recruit Wnt Ligands Required for Self-Renewal We previously described a system of a localized Wnt3a bead which recapitulates a niche signal essential for self-renewal and oriented asymmetric cell division (ACD) of single ESCs (20). Wnt5a, also produced by TSCs, cannot activate the Wnt/β-catenin pathway in ESCs (20). Importantly, Wnt5a beads do not induce ACD in ESCs (20). Using this Wnt-bead approach, we aimed to investigate the mechanisms by which ESCs interact with localized niche signals. We incubated single cells in close proximity to Wnt3a beads or Wnt5a beads (SI Appendix, Fig. S1D) and monitored initial cell-bead contact by live imaging. Primary observations revealed that ESCs utilize thin cytonemes to contact the bead and can react by directing a larger cytoneme to recruit the bead to the plasma membrane (reactive interaction [RI]) to form a stable contact (Figs. 1D and 2A and Movie S3). Although Wnt5a has high protein sequence similarity to Wnt3a, our assay indicated a significantly higher proportion of reactive interactions when cytonemes encountered Wnt3a beads (76% RI) relative to Wnt5a beads (43% RI) (Fig. 1E). This suggests that ESC cytonemes selectively react to Wnt ligands required for self-renewal. Fig. 2. ESCs extend exploratory actin-based cytonemes that recruit localized Wnt signals to the plasma membrane. (A) Representative frames from time-lapse imaging of an ESC contacting a Wnt3a bead (black sphere) with a cytoneme. Time is expressed in minutes. (Scale bar, 10 μm.) Arrowheads indicate thin cytonemes; arrows indicate larger cytonemes used for Wnt-bead recruitment. (B) Scanning electron microscopy images of ESCs at various stages of interaction with the Wnt3a bead (white/gray spheres). White arrowhead indicates a thin cytoneme. (Scale bar, 5 μm.) (C) Deconvolved structural illumination images of ESCs stained with the live-cell cytoskeleton-staining reagents SiR-Actin (Top) and SiR-Tubulin (Bottom). Arrows indicate larger cytonemes; arrowheads indicate thin cytonemes. (Scale bars, 10 μm.) (D) Representative images of an ESC cytoneme contacting a Wnt3a bead (dashed purple circle or 3D-reconstructed purple sphere) stained with antibodies against LRP6 (cyan) and β-catenin (magenta) with DAPI (yellow). (Scale bar, 20 μm.) (E) Representative images of an ESC contacting an eGFP-expressing TSC (green, dashed edge) stained with antibodies against β-catenin (magenta) with DAPI (yellow). (Scale bar, 20 μm.) We also tested the reactivity of cytonemes to control beads—inactive Wnt3a beads (iWnt3a beads) treated with dithiothreitol (DTT) to break the disulfide bridges in Wnt ligands, thus disrupting protein tertiary structure to render it inactive (refs. 20 and 24 and SI Appendix, Fig. S1D)—or to uncoated beads. Our results indicate that the cytonemes are unable to react to iWnt3a beads or uncoated beads efficiently (31% and 20% RI, respectively; Fig. 1E). To further confirm the selectivity of the cytonemes, we exposed ESCs to beads coated with bovine serum albumin (BSA), a nonsignaling molecule that often adheres nonspecifically to cellular membranes. Here, only 34% of interactions were reactive (Fig. 1E), reduced like the other control beads. In summary, ESCs generate ligand-selective cytonemes to identify and recruit Wnt signals required for self-renewal. This ligand-based selectivity also governs the efficiency of stable ESC–TSC contacts and pairing. To determine how cytonemes achieve this dual functionality, we analyzed their composition and dynamics.

Discussion Cytoneme-mediated signaling is a means of highly specific paracrine signal transduction, allowing both signal amplitude and duration to be controlled with exquisite precision. Yamashita and colleagues identified, in male germline stem cells, microtubule-based protrusions that contain bone morphogenic protein (BMP) receptors and extend to the hub cells in Drosophila testis, thereby recruiting the ligand that is essential for their maintenance (37). Identification of cytonemes and understanding of their function in mammalian stem cells remain limited. Here, we show that ESCs use specialized cytonemes to distinguish between niche signals and preferentially select Wnt ligands promoting their self-renewal, such as those secreted by TSCs. After the recognition of the Wnt source, ESCs generate larger cytonemes to facilitate robust signaling and pairing with TSCs to promote synthetic embryogenesis. The multiplicity of secreted ligands and the difficulty of visualizing Wnt proteins in situ make it challenging to study how Wnt reception occurs in ESCs. To circumvent these issues, we utilized a reductionist approach of immobilizing purified Wnt ligands on microbeads, distributing them near single ESCs, and observing their interaction by time-lapse imaging. We found that ESCs generate actin-based cytonemes that contain Wnt receptors, which represent, on average, 60% of the total protrusions that ESCs form. After a Wnt source is detected, the bulk of the receptors and β-catenin polarizes toward the Wnt. A larger cytoneme enriched with Wnt receptors and AMPA/kainate receptor subunits form a stable contact with the Wnt source. Consequently, LRP6-AMPA/kainate receptor crosstalk is initiated and generates localized Ca2+ transients. This crosstalk is required to allow the reactive interaction, Wnt signaling, and ESC–TSC pairing that form the basis of cellular communication and spatial self-organization. The finding that ESCs utilize iGluR-containing cytonemes indicates a striking similarity to aspects of the neuronal synapse. Both systems show selective, directed cellular protrusions for the purposes of reinforcing a “correct” signal and leading to higher-order spatial signaling and organization. Recent comprehensive investigations have demonstrated that components of the pre- and postsynapse are essential for cytoneme-mediated paracrine signaling in the Drosophila air sac primordium (28) and that neurotransmitter pathway components, including glutamatergic receptors, are present in Ctenophore during embryogenesis (38). These findings, and our own data, may suggest that the neuronal synapse shares a common ancestor with glutamatergic cytonemes used for cell–cell signaling, illustrating an evolutionarily conserved method for spatial organization of cells. Intracellular Ca2+ stores and components of the Wnt pathway regulate cytoneme formation. Specifically, components of the Wnt pathway control the number of cytonemes, their length, and their selectivity to the ligand. LRP6, but not LRP5, regulates the length of the cytoneme. This may be attributed to motifs in the N terminal of LRP6 that can bind to regulators of actin filaments and promote their elongation, branching, and dynamics (39⇓–41). Similarly, DVL2 has been shown to interact with actin regulators (42). DVL2KO produces fewer and shorter cytonemes in comparison to all other studied cell lines. We found that, as a Wnt-pathway effector, β-catenin levels are inversely correlated with the number of cytonemes. While the absence of β-catenin leads to an increase in the number of cytonemes, higher levels of β-catenin, such as those reported in Dvl2KO, repress cytoneme formation and consequently produce fewer and shorter cytonemes than WT. Considering all evidence, we showed that Wnt pathway components in ESCs not only are required for signal reception and transduction but also determine the efficiency of scanning the environment to locate self-renewal signals. In summary, we identified specialized cytonemes that form the basis of ESC–TSC communication that is fundamental to morphogenesis. The minimal approach of single ESCs interacting with immobilized niche ligands provides unique insights into the mechanisms of contact-mediated signaling. We envision that mechanisms identified in this study could be applied to adult stem cells and niches where the Wnt/β-catenin pathway is required for self-renewal.

Materials and Methods Details of the protocols for culture of ESCs and TSCs, generation and characterization of knockout ESC and reporter cell lines, purification and immobilization of Wnt proteins, conditions of cell imaging, image analysis, electron microscopy, flow cytometry, and statistical analyses performed can be found in SI Appendix. Data and Materials Availability. All additional data and information are included in SI Appendix as figures, additional legends, and references.

Acknowledgments We thank Huang and Kornberg for providing us with the manuscript of their 2019 Science article prior to publication. We thank the Nikon Imaging Centre at King’s College London for help with light microscopy. We thank Dr Mathieu Fréchin and Nanolive for their help in generating the 3D hologram tomography. We thank Dr. Kathy Niakan and Dr. Niwa Ali for constructive criticism of the manuscript. We acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre award to the Guy’s & St Thomas National Health Service Foundation Trust in partnership with King’s College London and the King’s College Hospital NHS Foundation Trust. This work was supported in part by a Sir Henry Dale Fellowship (102513/Z/13/Z) to S.J.H.

Footnotes Author contributions: S.J. and S.J.H. designed research; S.J., C.L.G., J.L.A.S., T.-J.T., J.R., and S.J.H. performed research; S.J., J.R., and S.J.H. contributed new reagents; S.J., C.L.G., J.L.A.S., T.-J.T., J.R., and S.J.H. analyzed data; and S.J. and S.J.H. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

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