Mechanical regulation of early vertebrate embryogenesis.

Embryonic cells grow in environments that provide a plethora of physical cues, including mechanical forces that shape the development of the entire embryo. Despite their prevalence, the role of these forces in embryonic development and their integration with chemical signals have been mostly neglected, and scrutiny in modern molecular embryology tilted, instead, towards the dissection of molecular pathways involved in cell fate determination and patterning. It is now possible to investigate how mechanical signals induce downstream genetic regulatory networks to regulate key developmental processes in the embryo. Here, we review the insights into mechanical control of early vertebrate development, including the role of forces in tissue patterning and embryonic axis formation. We also highlight recent in vitro approaches using individual embryonic stem cells and self-organizing multicellular models of human embryos, which have been instrumental in expanding our understanding of how mechanics tune cell fate and cellular rearrangements during human embryonic development.

The BMP inhibitor Coco reactivates breast cancer cells at lung metastatic sites.

The mechanistic underpinnings of metastatic dormancy and reactivation are poorly understood. A gain-of-function cDNA screen reveals that Coco, a secreted antagonist of TGF-ß ligands, induces dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicate that Coco exerts this effect by blocking lung-derived BMP ligands. Whereas Coco enhances the manifestation of traits associated with cancer stem cells, BMP signaling suppresses it. Coco induces a discrete gene expression signature, which is strongly associated with metastatic relapse to the lung, but not to the bone or brain in patients. Experiments in mouse models suggest that these latter organs contain niches devoid of bioactive BMP. These findings reveal that metastasis-initiating cells need to overcome organ-specific antimetastatic signals in order to undergo reactivation.

Mechanisms underlying WNT-mediated priming of human embryonic stem cells.

Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context-dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs). Our previous work demonstrated that during early stages of hESC differentiation, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening but, instead, acts by inducing the expression of the SMAD2 co-factor EOMES. Expression of EOMES is sufficient to replace WNT upstream of activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.

Huntingtin CAG expansion impairs germ layer patterning in synthetic human 2D gastruloids through polarity defects.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFß signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFß receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids.

High resolution dynamic ultrasound atlas of embryonic and fetal development of the common marmoset.

PURPOSE: The common marmoset (Callithrix jacchus) provides an ideal model to study early development of primates, and an in vivo platform to validate conclusions from in vitro studies of human embryos and embryo models. Currently, however, no established staging atlas of marmoset embryonic development exists. Using high-resolution, longitudinal ultrasound scans on live pregnant marmosets, we present the first dynamic in vivo imaging of entire primate gestation beginning with attachment until the last day before birth. METHODS: Our study unveils the first dynamic images of an in vivo attached mammalian embryo developing in utero, and the intricacies of the delayed development period unique to the common marmoset amongst primates, revealing a window for somatic interventions. RESULTS: Established obstetric and embryologic measurements for each scan were used comparatively with the standardized Carnegie staging of human development to highlight similarities and differences. Our study also allows for tracking the development of major organs. We focus on the ontogeny of the primate heart and brain. Finally, input ultrasound images were used to train deep neural networks to accurately determine the gestational age. All our ultrasounds and staging data recording are posted online so that the atlas can be used as a community resource toward monitoring and managing marmoset breeding colonies. CONCLUSION: The temporal and spatial resolution of ultrasound achieved in this study demonstrates the promise of noninvasive imaging in the marmoset for the in vivo study of primate-specific aspects of embryonic and fetal development.

Synthetic by design: Exploiting tissue self-organization to explore early human embryology.

Recent advances in synthetic human embryology has provided a previously inexistent molecular portrait of human development. Models of synthetic human embryonic tissues capitalize on the self-organizing capabilities of human embryonic stem cells when they are cultured on biomimetic conditions that simulate in vivo human development. In this Review, we discuss these models and how they have shed light on the early stages of human development including amniotic sac development, gastrulation and neurulation. We discuss the mechanisms underlying the molecular logic of embryonic tissue self-organization that have been dissected using synthetic models of human embryology and explore future challenges in the field. Geared with technological advances in bioengineering, high resolution gene expression and imaging tools, these models are set to transform our understanding of the mechanistic basis of embryonic tissue self-organization during human development and how they may go awry in disease.

A wave of WNT signaling balanced by secreted inhibitors controls primitive streak formation in micropattern colonies of human embryonic stem cells.

Long-range signaling by morphogens and their inhibitors define embryonic patterning yet quantitative data and models are rare, especially in humans. Here, we use a human embryonic stem cell micropattern system to model formation of the primitive streak (PS) by WNT. In the pluripotent state, E-cadherin (E-CAD) transduces boundary forces to focus WNT signaling to the colony border. Following application of WNT ligand, E-CAD mediates a front or wave of epithelial-to-mesenchymal (EMT) conversion analogous to PS extension in an embryo. By knocking out the secreted WNT inhibitors active in our system, we show that DKK1 alone controls the extent and duration of patterning. The NODAL inhibitor cerberus 1 acts downstream of WNT to refine the endoderm versus mesoderm fate choice. Our EMT wave is a generic property of a bistable system with diffusion and we present a single quantitative model that describes both the wave and our knockout data.

Mapping cell migrations and fates in a gastruloid model to the human primitive streak.

Although fate maps of early embryos exist for nearly all model organisms, a fate map of the gastrulating human embryo remains elusive. Here, we use human gastruloids to piece together a rudimentary fate map for the human primitive streak (PS). This is possible because differing levels of BMP, WNT and NODAL lead to self-organization of gastruloids into homogenous subpopulations of endoderm and mesoderm, and comparative analysis of these gastruloids, together with the fate map of the mouse embryo, allows the organization of these subpopulations along an anterior-posterior axis. We also developed a novel cell tracking technique that detected robust fate-dependent cell migrations in our gastruloids comparable with those found in the mouse embryo. Taken together, our fate map and recording of cell migrations provides a first coarse view of what the human PS may resemble in vivo.

Self-organization of the in vitro attached human embryo.

Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy.

Chromosomal instability during neurogenesis in Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by expansion of CAG repeats in the Huntingtin gene (HTT). Neither its pathogenic mechanisms nor the normal functions of HTT are well understood. To model HD in humans, we engineered a genetic allelic series of isogenic human embryonic stem cell (hESC) lines with graded increases in CAG repeat length. Neural differentiation of these lines unveiled a novel developmental HD phenotype: the appearance of giant multinucleated telencephalic neurons at an abundance directly proportional to CAG repeat length, generated by a chromosomal instability and failed cytokinesis over multiple rounds of DNA replication. We conclude that disrupted neurogenesis during development is an important, unrecognized aspect of HD pathogenesis. To address the function of normal HTT protein we generated HTT+/- and HTT-/- lines. Surprisingly, the same phenotype emerged in HTT-/- but not HTT+/- lines. We conclude that HD is a developmental disorder characterized by chromosomal instability that impairs neurogenesis, and that HD represents a genetic dominant-negative loss of function, contrary to the prevalent gain-of-toxic-function hypothesis. The consequences of developmental alterations should be considered as a new target for HD therapies.

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis.

Cells have an intrinsic ability to self-assemble and self-organize into complex and functional tissues and organs. By taking advantage of this ability, embryoids, organoids and gastruloids have recently been generated in vitro, providing a unique opportunity to explore complex embryological events in a detailed and highly quantitative manner. Here, we examine how such approaches are being used to answer fundamental questions in embryology, such as how cells self-organize and assemble, how the embryo breaks symmetry, and what controls timing and size in development. We also highlight how further improvements to these exciting technologies, based on the development of quantitative platforms to precisely follow and measure subcellular and molecular events, are paving the way for a more complete understanding of the complex events that help build the human embryo.

Coco is a dual activity modulator of TGFß signaling.

The TGFß signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFß ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFß1 signaling. Coco synergizes with TGFß1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFß1 and enhances TGFß1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFß signaling.

A method to recapitulate early embryonic spatial patterning in human embryonic stem cells.

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

Huntingtin is required for ciliogenesis and neurogenesis during early Xenopus development.

Huntington's Disease (HD) is a neurodegenerative disorder that results from the abnormal expansion of poly-glutamine (polyQ) repeats in the Huntingtin (HTT) gene. Although HTT has been linked to a variety of cellular events, it is still not clear what the physiological functions of the protein are. Because of its critical role during mouse embryonic mouse development, we investigated the functions of Htt during early Xenopus embryogenesis. We find that reduction of Htt levels affects cilia polarity and function and causes whole body paralysis. Moreover, Htt loss of function leads to abnormal development of trigeminal and motor neurons. Interestingly, these phenotypes are partially rescued by either wild-type or expanded HTT. These results show that the Htt activity is required for normal embryonic development, and highlight the usefulness of the Xenopus system for investigating proteins involved in human diseases.

Coco regulates dorsoventral specification of germ layers via inhibition of TGFß signalling.

One of the earliest steps in embryonic development is the specification of the germ layers, the subdivision of the blastula embryo into endoderm, mesoderm and ectoderm. Maternally expressed members of the Transforming Growth Factor ß (TGFß) family influence all three germ layers; the ligands are required to induce endoderm and mesoderm, whereas inhibitors are required for formation of the ectoderm. Here, we demonstrate a vital role for maternal Coco, a secreted antagonist of TGFß signalling, in this process. We show that Coco is required to prevent Activin and Nodal signals in the dorsal marginal side of the embryo from invading the prospective ectoderm, thereby restricting endoderm- and mesoderm-inducing signals to the vegetal and marginal zones of the pre-gastrula Xenopus laevis embryo.

APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.

Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5-two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of beta-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.

miR-373 is regulated by TGFß signaling and promotes mesendoderm differentiation in human Embryonic Stem Cells.

MicroRNAs (miRNAs) belonging to the evolutionary conserved miR-302 family play important functions in Embryonic Stem Cells (ESCs). The expression of some members, such as the human miR-302 and mouse miR-290 clusters, is regulated by ESC core transcription factors. However, whether miRNAs act downstream of signaling pathways involved in human ESC pluripotency remains unknown. The maintenance of pluripotency in hESCs is under the control of the TGFß pathway. Here, we show that inhibition of the Activin/Nodal branch of this pathway affects the expression of a subset of miRNAs in hESCs. Among them, we found miR-373, a member of the miR-302 family. Proper levels of miR-373 are crucial for the maintenance of hESC pluripotency, since its overexpression leads to differentiation towards the mesendodermal lineage. Among miR-373 predicted targets, involved in TGFß signaling, we validated the Nodal inhibitor Lefty. Our work suggests a crucial role for the interplay between miRNAs and signaling pathways in ESCs.

Huntingtin protein is essential for mitochondrial metabolism, bioenergetics and structure in murine embryonic stem cells.

Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntington's disease (HD), while htt(-/-) mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, we compared more than 3700 compounds in three syngeneic mouse embryonic stem cell (mESC) lines: htt(-/-), extended poly-Q (Htt-Q140/7), and wild-type mESCs (Htt-Q7/7) using untargeted metabolite profiling. While Htt-Q140/7 cells did not show major differences in cellular bioenergetics, we find extensive metabolic aberrations in htt(-/-) mESCs, including (i) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (ii) near-maximal glycolysis, with little or no glycolytic reserve; (iii) marked ketogenesis; (iv) depletion of intracellular NTPs; (v) accelerated purine biosynthesis and salvage; and (vi) loss of mitochondrial structural integrity. Together, our findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt(-/-) early embryonic lethality.

A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation.

Multiple levels of control are in play to regulate pluripotency and differentiation in human embryonic stem cells (hESCs). At the transcriptional level, the core factors OCT4, NANOG and SOX2 form a positive autoregulatory loop that is pivotal for maintaining the undifferentiated state. At the post-transcriptional level, microRNAs (miRNAs) belonging to the miR-302 family are emerging as key players in the control of proliferation and cell fate determination during differentiation. Here, we show that the transcriptional factors OCT4 and NR2F2 (COUP-TFII) and the miRNA miR-302 are linked in a regulatory circuitry that critically regulate both pluripotency and differentiation in hESCs. In the undifferentiated state, both OCT4 and the OCT4-induced miR-302 directly repress NR2F2 at the transcriptional and post-transcriptional level, respectively. Conversely, NR2F2 directly inhibits OCT4 during differentiation, triggering a positive feedback loop for its own expression. In addition, we show that regulation of NR2F2 activity itself relies on alternative splicing and transcriptional start site choice to generate a full-length transcriptionally active isoform and shorter variants, which enhance the activity of the long isoform. During hESC differentiation, NR2F2 is first detected at the earliest steps of neural induction and thus is among the earliest human embryonic neural markers. Finally, our functional analysis points to a crucial role for NR2F2 in the activation of neural genes during early differentiation in humans. These findings introduce a new molecular player in the context of early embryonic stem cell state and cell fate determination in humans.

A taste of TGFß in Tuscany.

The recent FASEB Summer Research Conference entitled 'The TGFß Superfamily: Signaling in Development and Disease' was held in August, 2011 in the spectacular setting of Il Ciocco, Lucca, amidst the olive trees in Tuscany, Italy. The organizers assembled an amazing forum, which included 53 speakers and 67 poster presentations from laboratories around the world, to showcase recent advances made in our understanding of the transforming growth factor-ß (TGFß) signaling pathway.