Loss of Notch signaling in skeletal stem cells enhances bone formation with aging.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

Genetic models for lineage tracing in musculoskeletal development, injury, and healing.

Musculoskeletal development and later post-natal homeostasis are highly dynamic processes, marked by rapid structural and functional changes across very short periods of time. Adult anatomy and physiology are derived from pre-existing cellular and biochemical states. Consequently, these early developmental states guide and predict the future of the system as a whole. Tools have been developed to mark, trace, and follow specific cells and their progeny either from one developmental state to the next or between circumstances of health and disease. There are now many such technologies alongside a library of molecular markers which may be utilized in conjunction to allow for precise development of unique cell 'lineages'. In this review, we first describe the development of the musculoskeletal system beginning as an embryonic germ layer and at each of the key developmental stages that follow. We then discuss these structures in the context of adult tissues during homeostasis, injury, and repair. Special focus is given in each of these sections to the key genes involved which may serve as markers of lineage or later in post-natal tissues. We then finish with a technical assessment of lineage tracing and the techniques and technologies currently used to mark cells, tissues, and structures within the musculoskeletal system.

Hox genes are crucial regulators of periosteal stem cell identity.

Periosteal stem and progenitor cells (PSPCs) are major contributors to bone maintenance and repair. Deciphering the molecular mechanisms that regulate their function is crucial for the successful generation and application of future therapeutics. Here, we pinpoint Hox transcription factors as necessary and sufficient for periosteal stem cell function. Hox genes are transcriptionally enriched in periosteal stem cells and their overexpression in more committed progenitors drives reprogramming to a naïve, self-renewing stem cell-like state. Crucially, individual Hox family members are expressed in a location-specific manner and their stem cell-promoting activity is only observed when the Hox gene is matched to the anatomical origin of the PSPC, demonstrating a role for the embryonic Hox code in adult stem cells. Finally, we demonstrate that Hoxa10 overexpression partially restores the age-related decline in fracture repair. Together, our data highlight the importance of Hox genes as key regulators of PSPC identity in skeletal homeostasis and repair.

Modulating the systemic and local adaptive immune response after fracture improves bone regeneration during aging.

Tissue injury leads to the well-orchestrated mobilization of systemic and local innate and adaptive immune cells. During aging, immune cell recruitment is dysregulated, resulting in an aberrant inflammatory response that is detrimental for successful healing. Here, we precisely define the systemic and local immune cell response after femur fracture in young and aging mice and identify increased toll-like receptor signaling as a potential culprit for the abnormal immune cell recruitment observed in aging animals. Myd88, an upstream regulator of TLR-signaling lies at the core of this aging phenotype, and local treatment of femur fractures with a Myd88 antagonist in middle-aged mice reverses the aging phenotype of impaired fracture healing, thus offering a promising therapeutic target that could overcome the negative impact of aging on bone regeneration.

Quantitative analysis of signaling responses during mouse primordial germ cell specification.

During early mammalian development, the pluripotent cells of the embryo are exposed to a combination of signals that drive exit from pluripotency and germ layer differentiation. At the same time, a small population of pluripotent cells give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. Despite the importance of PGCs, it remains unclear how they are first segregated from the soma, and if this involves distinct responses to their signaling environment. To investigate this question, we mapped BMP, MAPK and WNT signaling responses over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution. We showed that, in the mouse embryo, early PGCs exhibit lower BMP and MAPK responses compared to neighboring extraembryonic mesoderm cells, suggesting the emergence of distinct signaling regulatory mechanisms in the germline versus soma. In contrast, PGCs and somatic cells responded comparably to WNT, indicating that this signal alone is not sufficient to promote somatic differentiation. Finally, we investigated the requirement of a BMP response for these cell fate decisions. We found that cell lines with a mutation in the BMP receptor (Bmpr1a-/-), which exhibit an impaired BMP signaling response, can efficiently generate PGC-like cells revealing that canonical BMP signaling is not cell autonomously required to direct PGC-like differentiation.

The transcription factor Rreb1 regulates epithelial architecture, invasiveness, and vasculogenesis in early mouse embryos.

Ras-responsive element-binding protein 1 (Rreb1) is a zinc-finger transcription factor acting downstream of RAS signaling. Rreb1 has been implicated in cancer and Noonan-like RASopathies. However, little is known about its role in mammalian non-disease states. Here, we show that Rreb1 is essential for mouse embryonic development. Loss of Rreb1 led to a reduction in the expression of vasculogenic factors, cardiovascular defects, and embryonic lethality. During gastrulation, the absence of Rreb1 also resulted in the upregulation of cytoskeleton-associated genes, a change in the organization of F-ACTIN and adherens junctions within the pluripotent epiblast, and perturbed epithelial architecture. Moreover, Rreb1 mutant cells ectopically exited the epiblast epithelium through the underlying basement membrane, paralleling cell behaviors observed during metastasis. Thus, disentangling the function of Rreb1 in development should shed light on its role in cancer and other diseases involving loss of epithelial integrity.

Spatially Organized Differentiation of Mouse Pluripotent Stem Cells on Micropatterned Surfaces.

Pluripotent stem cells (PSCs) are the in vitro counterpart of the pluripotent epiblast of the mammalian embryo with the capacity to generate all cell types of the adult organism. During development, the three definitive germ layers are specified and simultaneously spatially organized. In contrast, differentiating PSCs tend to generate cell fates in a spatially disorganized manner. This has limited the in vitro study of specific cell-cell interactions and patterning mechanisms that occur in vivo. Here we describe a protocol to differentiate mouse PSCs in a spatially organized manner on micropatterned surfaces. Micropatterned chips comprise many colonies of uniform size and geometry facilitating a robust quantitative analysis of patterned fate specification. Furthermore, multiple factors may be simultaneously manipulated with temporal accuracy to probe the dynamic interactions regulating these processes. The micropattern system is scalable, providing a valuable tool to generate material for large-scale analysis and biochemical experiments that require substantial amounts of starting material, difficult to obtain from early embryos.

Signaling regulation during gastrulation: Insights from mouse embryos and in vitro systems.

Gastrulation is the process whereby cells exit pluripotency and concomitantly acquire and pattern distinct cell fates. This is driven by the convergence of WNT, BMP, Nodal and FGF signals, which are tightly spatially and temporally controlled, resulting in regional and stage-specific signaling environments. The combination, level and duration of signals that a cell is exposed to, according its position within the embryo and the developmental time window, dictates the fate it will adopt. The key pathways driving gastrulation exhibit complex interactions, which are difficult to disentangle in vivo due to the complexity of manipulating multiple signals in parallel with high spatiotemporal resolution. Thus, our current understanding of the signaling dynamics regulating gastrulation is limited. In vitro stem cell models have been established, which undergo organized cellular differentiation and patterning. These provide amenable, simplified, deconstructed and scalable models of gastrulation. While the foundation of our understanding of gastrulation stems from experiments in embryos, in vitro systems are now beginning to reveal the intricate details of signaling regulation. Here we discuss the current state of knowledge of the role, regulation and dynamic interaction of signaling pathways that drive mouse gastrulation.

Publisher Correction: TGF-ß orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

TGF-ß orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1.

Epithelial-to-mesenchymal transitions (EMTs) are phenotypic plasticity processes that confer migratory and invasive properties to epithelial cells during development, wound-healing, fibrosis and cancer1-4. EMTs are driven by SNAIL, ZEB and TWIST transcription factors5,6 together with microRNAs that balance this regulatory network7,8. Transforming growth factor ß (TGF-ß) is a potent inducer of developmental and fibrogenic EMTs4,9,10. Aberrant TGF-ß signalling and EMT are implicated in the pathogenesis of renal fibrosis, alcoholic liver disease, non-alcoholic steatohepatitis, pulmonary fibrosis and cancer4,11. TGF-ß depends on RAS and mitogen-activated protein kinase (MAPK) pathway inputs for the induction of EMTs12-19. Here we show how these signals coordinately trigger EMTs and integrate them with broader pathophysiological processes. We identify RAS-responsive element binding protein 1 (RREB1), a RAS transcriptional effector20,21, as a key partner of TGF-ß-activated SMAD transcription factors in EMT. MAPK-activated RREB1 recruits TGF-ß-activated SMAD factors to SNAIL. Context-dependent chromatin accessibility dictates the ability of RREB1 and SMAD to activate additional genes that determine the nature of the resulting EMT. In carcinoma cells, TGF-ß-SMAD and RREB1 directly drive expression of SNAIL and fibrogenic factors stimulating myofibroblasts, promoting intratumoral fibrosis and supporting tumour growth. In mouse epiblast progenitors, Nodal-SMAD and RREB1 combine to induce expression of SNAIL and mesendoderm-differentiation genes that drive gastrulation. Thus, RREB1 provides a molecular link between RAS and TGF-ß pathways for coordinated induction of developmental and fibrogenic EMTs. These insights increase our understanding of the regulation of epithelial plasticity and its pathophysiological consequences in development, fibrosis and cancer.

Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-ß signaling.

TGF-ß receptors phosphorylate SMAD2 and SMAD3 transcription factors, which then form heterotrimeric complexes with SMAD4 and cooperate with context-specific transcription factors to activate target genes. Here we provide biochemical and structural evidence showing that binding of SMAD2 to DNA depends on the conformation of the E3 insert, a structural element unique to SMAD2 and previously thought to render SMAD2 unable to bind DNA. Based on this finding, we further delineate TGF-ß signal transduction by defining distinct roles for SMAD2 and SMAD3 with the forkhead pioneer factor FOXH1 as a partner in the regulation of differentiation genes in mouse mesendoderm precursors. FOXH1 is prebound to target sites in these loci and recruits SMAD3 independently of TGF-ß signals, whereas SMAD2 remains predominantly cytoplasmic in the basal state and set to bind SMAD4 and join SMAD3:FOXH1 at target promoters in response to Nodal TGF-ß signals. The results support a model in which signal-independent binding of SMAD3 and FOXH1 prime mesendoderm differentiation gene promoters for activation, and signal-driven SMAD2:SMAD4 binds to promoters that are preloaded with SMAD3:FOXH1 to activate transcription.

A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice.

The FGF/ERK signaling pathway is highly conserved throughout evolution and plays fundamental roles during embryonic development and in adult organisms. While a plethora of expression data exists for ligands, receptors and pathway regulators, we know little about the spatial organization or dynamics of signaling in individual cells within populations. To this end we developed a transcriptional readout of FGF/ERK activity by targeting a histone H2B-linked Venus fluorophore to the endogenous locus of Spry4, an early pathway target, and generated Spry4H2B-Venus embryonic stem cells (ESCs) and a derivative mouse line. The Spry4H2B-Venus reporter was heterogeneously expressed within ESC cultures and responded to FGF/ERK signaling manipulation. In vivo, the Spry4H2B-Venus reporter recapitulated the expression pattern of Spry4 and localized to sites of known FGF/ERK activity including the inner cell mass of the pre-implantation embryo and the limb buds, somites and isthmus of the post-implantation embryo. Additionally, we observed highly localized reporter expression within adult organs. Genetic and chemical disruption of FGF/ERK signaling, in vivo in pre- and post-implantation embryos, abrogated Venus expression establishing the reporter as an accurate signaling readout. This tool will provide new insights into the dynamics of the FGF/ERK signaling pathway during mammalian development.

Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning.

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species.

Four simple rules that are sufficient to generate the mammalian blastocyst.

Early mammalian development is both highly regulative and self-organizing. It involves the interplay of cell position, predetermined gene regulatory networks, and environmental interactions to generate the physical arrangement of the blastocyst with precise timing. However, this process occurs in the absence of maternal information and in the presence of transcriptional stochasticity. How does the preimplantation embryo ensure robust, reproducible development in this context? It utilizes a versatile toolbox that includes complex intracellular networks coupled to cell-cell communication, segregation by differential adhesion, and apoptosis. Here, we ask whether a minimal set of developmental rules based on this toolbox is sufficient for successful blastocyst development, and to what extent these rules can explain mutant and experimental phenotypes. We implemented experimentally reported mechanisms for polarity, cell-cell signaling, adhesion, and apoptosis as a set of developmental rules in an agent-based in silico model of physically interacting cells. We find that this model quantitatively reproduces specific mutant phenotypes and provides an explanation for the emergence of heterogeneity without requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells' competence of fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively by manipulating embryos in vitro. Based on these observations, we conclude that the minimal set of rules enables the embryo to experiment with stochastic gene expression and could provide the robustness necessary for the evolutionary diversification of the preimplantation gene regulatory network.

The many faces of Pluripotency: in vitro adaptations of a continuum of in vivo states.

Pluripotency defines the propensity of a cell to differentiate into, and generate, all somatic, as well as germ cells. The epiblast of the early mammalian embryo is the founder population of all germ layer derivatives and thus represents the bona fide in vivo pluripotent cell population. The so-called pluripotent state spans several days of development and is lost during gastrulation as epiblast cells make fate decisions towards a mesoderm, endoderm or ectoderm identity. It is now widely recognized that the features of the pluripotent population evolve as development proceeds from the pre- to post-implantation period, marked by distinct transcriptional and epigenetic signatures. During this period of time epiblast cells mature through a continuum of pluripotent states with unique properties. Aspects of this pluripotent continuum can be captured in vitro in the form of stable pluripotent stem cell types. In this review we discuss the continuum of pluripotency existing within the mammalian embryo, using the mouse as a model, and the cognate stem cell types that can be derived and propagated in vitro. Furthermore, we speculate on embryonic stage-specific characteristics that could be utilized to identify novel, developmentally relevant, pluripotent states.

Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency.

Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i) or with knockout serum replacement (KOSR), but not serum, can generate high-level chimeras regardless of how these cells were derived. ESCs cultured in these conditions showed a transcriptional correlation with early pre-implantation embryos (E1.5-E3.5) and contributed to development from the 2-cell stage. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition.

LIF supports primitive endoderm expansion during pre-implantation development.

Embryonic stem cells (ESCs) are pluripotent cell lines that can be maintained indefinitely in an early developmental state. ESC culture conditions almost always require the cytokine LIF to maintain self-renewal. As ESCs are not homogeneous but contain multiple populations reminiscent of the blastocyst, identifying the target cells of LIF is necessary to understand the propagation of pluripotency. We recently found that LIF acts under self-renewing conditions to stimulate the fraction of ESCs that express extraembryonic markers, but has little impact on pluripotent gene expression. Here, we report that LIF has two distinct roles: it blocks early epiblast (Epi) differentiation, and it supports the expansion of primitive endoderm (PrE)-primed ESCs and PrE in vivo. We find that activation of JAK/STAT signalling downstream of LIF occurs initially throughout the pre-implantation embryo, but later marks the PrE. Moreover, the addition of LIF to cultured embryos increases the GATA6(+) PrE population, whereas inhibition of JAK/STAT signalling reduces both NANOG(+) epiblast and GATA6(+) PrE. The reduction of the NANOG(+) Epi might be explained by its precocious differentiation to later Epi derivatives, whereas the increase in PrE is mediated both by an increase in proliferation and inhibition of PrE apoptosis that is normally triggered in embryos with an excess of GATA6(+) cells. Thus, it appears that the relative size of the PrE is determined by the number of LIF-producing cells in the embryo. This suggests a mechanism by which the embryo adjusts the relative ratio of the primary lineages in response to experimental manipulation.

The molecular underpinnings of totipotency.

Embryonic stem (ES) cells are characterized by their functional potency and capacity to self-renew in culture. Historically, ES cells have been defined as pluripotent, able to make the embryonic but not the extraembryonic lineages (such as the yolk sac and the placenta). The functional capacity of ES cells has been judged based on their ability to contribute to all somatic lineages when they are introduced into an embryo. However, a number of recent reports have suggested that under certain conditions, ES cells, and other reprogrammed cell lines, can also contribute to the extraembryonic lineages and, therefore, can be said to be totipotent. Here, we consider the molecular basis for this totipotent state, its transcriptional signature and the signalling pathways that define it.

Survival of the fattest.

Experiments on the social amoeba Dictyostelium discoideum show that the origins of lineage bias in this system lie in the nutritional history of individual cells. Clues to the molecular basis for this process suggest similar forces may be at work in early mammalian development.

Totipotent embryonic stem cells arise in ground-state culture conditions.

Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.