Time-aligned hourglass gastrulation models in rabbit and mouse.

The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals.

The intrinsic and extrinsic effects of TET proteins during gastrulation.

Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.

A single-embryo, single-cell time-resolved model for mouse gastrulation.

Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.

H3K27me3-mediated silencing of structural genes is required for zebrafish heart regeneration.

Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3K27M-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.

Notch signaling regulates cardiomyocyte proliferation during zebrafish heart regeneration.

The human heart's failure to replace ischemia-damaged myocardium with regenerated muscle contributes significantly to the worldwide morbidity and mortality associated with coronary artery disease. Remarkably, certain vertebrate species, including the zebrafish, achieve complete regeneration of amputated or injured myocardium through the proliferation of spared cardiomyocytes. Nonetheless, the genetic and cellular determinants of natural cardiac regeneration remain incompletely characterized. Here, we report that cardiac regeneration in zebrafish relies on Notch signaling. Following amputation of the zebrafish ventricular apex, Notch receptor expression becomes activated specifically in the endocardium and epicardium, but not the myocardium. Using a dominant negative approach, we discovered that suppression of Notch signaling profoundly impairs cardiac regeneration and induces scar formation at the amputation site. We ruled out defects in endocardial activation, epicardial activation, and dedifferentiation of compact myocardial cells as causative for the regenerative failure. Furthermore, coronary endothelial tubes, which we lineage traced from preexisting endothelium in wild-type hearts, formed in the wound despite the myocardial regenerative failure. Quantification of myocardial proliferation in Notch-suppressed hearts revealed a significant decrease in cycling cardiomyocytes, an observation consistent with a noncell autonomous requirement for Notch signaling in cardiomyocyte proliferation. Unexpectedly, hyperactivation of Notch signaling also suppressed cardiomyocyte proliferation and heart regeneration. Taken together, our data uncover the exquisite sensitivity of regenerative cardiomyocyte proliferation to perturbations in Notch signaling.

Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network.

BACKGROUND: Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors. RESULTS: Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other¿s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation. CONCLUSIONS: Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

LGN-dependent orientation of cell divisions in the dermomyotome controls lineage segregation into muscle and dermis.

The plane of cell divisions is pivotal for differential fate acquisition. Dermomyotome development provides an excellent system with which to investigate the link between these processes. In the central sheet of the early dermomyotome, single epithelial cells divide with a planar orientation. Here, we report that in the avian embryo, in addition to self-renewing, a subset of progenitors translocates into the myotome where they generate differentiated myocytes. By contrast, in the late epithelium, individual progenitors divide perpendicularly to produce both mitotic myoblasts and dermis. To examine whether spindle orientations influence fate segregation, early planar divisions were randomized and/or shifted to a perpendicular orientation by interfering with LGN function or by overexpressing inscuteable. Clones derived from single transfected cells exhibited an enhanced proportion of mixed dermomyotome/myotome progeny at the expense of `like' daughter cells in either domain. Loss of LGN or Gαi1 function in the late epithelium randomized otherwise perpendicular mitoses and favored muscle development at the expense of dermis. Hence, LGN-dependent early planar divisions are required for the proper allocation of progenitors into either dermomyotome or myotome, whereas late perpendicular divisions are necessary for the normal balance between muscle and dermis production.

Balanced gene dosage control rather than parental origin underpins genomic imprinting.

Mammalian parental imprinting represents an exquisite form of epigenetic control regulating the parent-specific monoallelic expression of genes in clusters. While imprinting perturbations are widely associated with developmental abnormalities, the intricate regional interplay between imprinted genes makes interpreting the contribution of gene dosage effects to phenotypes a challenging task. Using mouse models with distinct deletions in an intergenic region controlling imprinting across the Dlk1-Dio3 domain, we link changes in genetic and epigenetic states to allelic-expression and phenotypic outcome in vivo. This determined how hierarchical interactions between regulatory elements orchestrate robust parent-specific expression, with implications for non-imprinted gene regulation. Strikingly, flipping imprinting on the parental chromosomes by crossing genotypes of complete and partial intergenic element deletions rescues the lethality of each deletion on its own. Our work indicates that parental origin of an epigenetic state is irrelevant as long as appropriate balanced gene expression is established and maintained at imprinted loci.

Cell rearrangements during development of the somite and its derivatives.

The generation of somites, and the subsequent formation of their major derivatives, muscle-, cartilage-, dermis- and tendon-cell lineages, is tightly orchestrated and, to different extents, these are also mutually supporting processes. They involve complex and timely reorganizations of the paraxial mesoderm, such as multiple phases of epithelial-mesenchymal rearrangements and vice-versa, cellular movements and migrations, and modifications of both cell shape and cell cycle properties. These morphogenetic changes are triggered by local environmental signals and are tightly associated to a genetic program imparting cell-specific fates. Elucidating these signals and their downstream effectors, in addition to determining the state of specification of responsive cell subsets and that of single progenitors in the various domains, is only beginning.

Endocardial Notch Signaling Promotes Cardiomyocyte Proliferation in the Regenerating Zebrafish Heart through Wnt Pathway Antagonism.

Previous studies demonstrate that the regenerative zebrafish heart responds to injury by upregulating Notch receptors in the endocardium and epicardium. Moreover, global suppression of Notch activity following injury impairs cardiomyocyte proliferation and induces scarring. However, the lineage-specific requirements for Notch signaling and full array of downstream targets remain unidentified. Here, we demonstrate that inhibition of endocardial Notch signaling following ventricular amputation compromises cardiomyocyte proliferation and stimulates fibrosis. RNA sequencing uncovered reduced levels of two transcripts encoding secreted Wnt antagonists, Wif1 and Notum1b, in Notch-suppressed hearts. Like Notch receptors, wif1 and notum1b are induced following injury in the endocardium and epicardium. Small-molecule-mediated activation of Wnt signaling is sufficient to impair cardiomyocyte proliferation and induce scarring. Last, Wnt pathway suppression partially restored cardiomyocyte proliferation in hearts experiencing endocardial Notch inhibition. Taken together, our data demonstrate that Notch signaling supports cardiomyocyte proliferation by dampening myocardial Wnt activity during zebrafish heart regeneration.

Mechanisms of lineage segregation in the avian dermomyotome.

The somite and its intermediate derivatives, sclerotome and dermomyotome (DM), are composed of distinct subdomains based on lineage analysis and gene expression patterns. This sets the grounds for elucidating the mechanisms underlying differential cell specification and morphogenesis. By examining the in vivo roles of N-cadherin on discrete domains of the somitic epithelium at various times, our recent studies highlight the existence of a regional and temporal heterogeneity in cellular responsiveness. As examples of this assortment, we document a coupling between asymmetric cell division and fate segregation in the DM sheet, sequential effects of N-cadherin-mediated adhesion on early myogenic specification compared to later myofiber patterning, and a differential behavior of pioneer myoblasts compared to later myogenic waves.

Single cell transfection in chick embryos.

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Notch and bone morphogenetic protein differentially act on dermomyotome cells to generate endothelium, smooth, and striated muscle.

We address the mechanisms underlying generation of skeletal muscle, smooth muscle, and endothelium from epithelial progenitors in the dermomyotome. Lineage analysis shows that of all epithelial domains, the lateral region is the most prolific producer of smooth muscle and endothelium. Importantly, individual labeled lateral somitic cells give rise to only endothelial or mural cells (not both), and endothelial and mural cell differentiation is driven by distinct signaling systems. Notch activity is necessary for smooth muscle production while inhibiting striated muscle differentiation, yet it does not affect initial development of endothelial cells. On the other hand, bone morphogenetic protein signaling is required for endothelial cell differentiation and/or migration but inhibits striated muscle differentiation and fails to impact smooth muscle cell production. Hence, although different mechanisms are responsible for smooth muscle and endothelium generation, the choice to become smooth versus striated muscle depends on a single signaling system. Altogether, these findings underscore the spatial and temporal complexity of lineage diversification in an apparently homogeneous epithelium.

Medial pioneer fibers pattern the morphogenesis of early myoblasts derived from the lateral somite.

The first wave of myoblasts which constitutes the post-mitotic myotome stems from the medial epithelial somite. Whereas medial pioneers extend throughout the entire mediolateral myotome at cervical and limb levels, at flank regions they are complemented laterally by a population of early myoblasts emerging from the lateral epithelial somite. These myoblasts delaminate underneath the nascent dermomyotome and become post-mitotic. They are Myf5-positive but express MyoD and desmin only a day later while differentiating into fibers. Overexpression of Noggin in the lateral somite triggers their premature differentiation suggesting that lateral plate-BMP4 maintains them in an undifferentiated state. Moreover, directly accelerating their differentiation by MyoD overexpression prior to arrival of medial fibers, generates a severely mispatterned lateral myotome. This is in contrast to medial pioneers that have the capacity for self-organization. Furthermore, inhibiting differentiation of medial pioneers with dominant-negative MyoD also disrupts lateral myoblast patterning and differentiation. Thus, we propose that medial pioneers are needed for proper morphogenesis of the lateral population which is kept as undifferentiated mesenchyme by BMP4 until their arrival. In addition, medial pioneers also organize dermomyotome lip-derived fibers suggesting that they have a general role in patterning myotome development.

Differential effects of N-cadherin-mediated adhesion on the development of myotomal waves.

Myotomal fibers form by a first wave of pioneer myoblasts from the medial epithelial somite, and by a second wave from all four lips of the dermomyotome. Then, a third wave of mitotic progenitors colonizes the myotome, initially stemming from the extreme lips and, later, from the central dermomyotome sheet. In vitro studies have suggested that N-cadherin plays a role in myogenesis, but its role in vivo remains poorly understood. We find that during the growth phase of the dermomyotome sheet, when the orientation of mitotic spindles is parallel to the mediolateral extent of the epithelium, N-cadherin protein is inherited by both daughter cells. Prior to dermomyotome dissociation into dermis and muscle progenitors, when mitoses become perpendicularly oriented, N-cadherin remains associated only with the apical cell located in apposition to the myotome, generating molecular asymmetry between basal and apical progeny. Local gene missexpression confirms that N-cadherin-mediated adhesion is sufficient to promote myotome colonization, whereas its absence drives cells towards the subectodermal domain, hence coupling the asymmetric distribution of N-cadherin to a shift in mitotic orientation and to fate segregation. Site-directed electroporation to additional, discrete somite regions, further reveals that N-cadherin-mediated adhesion is necessary for maintaining the epithelial configuration of all dermomyotome domains while promoting the onset of Myod transcription and the translocation into the myotome of myofibers and/or of Pax-positive progenitors. By contrast, N-cadherin has no effect on migration or differentiation of the first wave of myotomal pioneers. Altogether, we show for the first time that the asymmetric localization of N-cadherin during mitosis indirectly influences fate segregation by differentially driving the allocation of progenitors to muscle versus dermal primordia, that the adhesive domain of N-cadherin maintains the integrity of the dermomyotome epithelium, which is necessary for myogenic specification, and that different molecular mechanisms underlie the establishment of pioneer and later myotomal waves.

Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates.

The dermomyotome develops into myotome and dermis. We previously showed that overall growth of the dermomyotome and myotome in the mediolateral direction occurs in a uniform pattern. While myofibers arise from all four dermomyotome lips, the dermis derives from both medial and lateral halves of the dermomyotome sheet. Here we mapped the fate of this epithelial sheet by analyzing cell types that arise from its central region. We found that these precursors give rise not only to dermis, as expected, but also to a population of proliferating progenitors in the myotome that maintain expression of PAX7, PAX3 and FREK. Given this dual fate, we asked whether single dermomyotome precursors generate both dermal and mitotic myoblast precursors, or alternatively, whether these cell types derive from distinct epithelial founders. Inovo clonal analysis revealed that single dermomyotome progenitors give rise to both derivatives. This is associated with a sharp change in the plane of cell division from the young epithelium, in which symmetrical divisions occur parallel to the mediolateral plane of the dermomyotome, to the dissociating dermomyotome, in which cell divisions become mostly perpendicular. Taken together with clonal analysis of the dermomyotome sheet, this suggests that a first stage of progenitor self-renewal, accounting for dermomyotomal expansion, is followed by fate segregation, which correlates with the observed shift in mitotic spindle orientation.

Coherent development of dermomyotome and dermis from the entire mediolateral extent of the dorsal somite.

We have previously shown that overall growth of the myotome in the mediolateral direction occurs in a coherent and uniform pattern. We asked whether development of the dermomyotome and resultant dermis follow a similar pattern or are, alternatively, controlled by restricted pools of stem cells driving directional growth. To this end, we studied cellular events that govern dermomyotome development and the regional origin of dermis. Measurements of cell proliferation, nuclear density and cellular rearrangements revealed that the developing dermomyotome can be subdivided in the transverse plane into three distinct and dynamic regions: medial, central and lateral, rather than simply into epaxial and hypaxial domains. To understand how these temporally and spatially restricted changes affect overall dermomyotome growth, lineage tracing with CM-DiI was performed. A proportional pattern of growth was measured along the entire epithelium, suggesting that mediolateral growth of the dermomyotome is coherent. Hence, they contrast with a stem cell view suggesting focal and inversely oriented sources of growth restricted to the medial and lateral edges. Consistent with this uniform mediolateral growth, lineage tracing experiments showed that the dermomyotome-derived dermis originates from progenitors that reside along the medial as well as the lateral halves of somites, and whose contribution to dermis is regionally restricted. Taken together, our results support the view that all derivatives of the dorsal somite (dermomyotome, myotome and dermis) keep a direct topographical relationship with their epithelial ascendants.