Proteostasis governs differential temperature sensitivity across embryonic cell types.

Embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. Phenotypes associated with environmental stress suggest that some cell types are more sensitive to stress than others, but the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single-cell RNA sequencing (RNA-seq) to identify cell types and molecular programs driving phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in developmental timing. The notochord is particularly sensitive to temperature, which we map to a specialized cell type: sheath cells. These cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and anatomic defects. Our study demonstrates that whole-animal single-cell RNA-seq can identify mechanisms for developmental robustness and pinpoint cell types that constitute key failure points.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Embryo-scale reverse genetics at single-cell resolution.

The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos1-4. A majority of these data, however, has been collected from wild-type embryos without an appreciation for the latent variation that is present in development. Here we present the 'zebrafish single-cell atlas of perturbed embryos': single-cell transcriptomic data from 1,812 individually resolved developing zebrafish embryos, encompassing 19 timepoints, 23 genetic perturbations and a total of 3.2 million cells. The high degree of replication in our study (eight or more embryos per condition) enables us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild-type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about early origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single-cell data from large numbers of individual embryos will enable mapping of the genetic dependencies of zebrafish cell types, while also addressing longstanding challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.

Identification of in vivo Hox13-binding sites reveals an essential locus controlling zebrafish brachyury expression.

During early embryogenesis, the vertebrate embryo extends from anterior to posterior because of the progressive addition of cells from a posteriorly localized neuromesodermal progenitor (NMp) population. An autoregulatory loop between Wnt and Brachyury/Tbxt is required for NMps to retain mesodermal potential and, hence, normal axis development. We recently showed that Hox13 genes help to support body axis formation and to maintain the autoregulatory loop, although the direct Hox13 target genes were unknown. Here, using a new method for identifying in vivo transcription factor-binding sites, we identified more than 500 potential Hox13 target genes in zebrafish. Importantly, we found two highly conserved Hox13-binding elements far from the tbxta transcription start site that also contain a conserved Tcf7/Lef1 (Wnt response) site. We show that the proximal of the two elements is sufficient to confer somitogenesis-stage expression to a tbxta promoter that, on its own, only drives NMp expression during gastrulation. Importantly, elimination of this proximal element produces shortened embryos due to aberrant formation of the most posterior somites. Our study provides a potential direct connection between Hox13 and regulation of the Wnt/Brachyury loop.

Hox13 genes are required for mesoderm formation and axis elongation during early zebrafish development.

The early vertebrate embryo extends from anterior to posterior due to the addition of neural and mesodermal cells from a neuromesodermal progenitor (NMp) population located at the most posterior end of the embryo. In order to produce mesoderm throughout this time, the NMps produce their own niche, which is high in Wnt and low in retinoic acid. Using a loss-of-function approach, we demonstrate here that the two most abundant Hox13 genes in zebrafish have a novel role in providing robustness to the NMp niche by working in concert with the niche-establishing factor Brachyury to allow mesoderm formation. Mutants lacking both hoxa13b and hoxd13a in combination with reduced Brachyury activity have synergistic posterior body defects, in the strongest case producing embryos with severe mesodermal defects that phenocopy brachyury null mutants. Our results provide a new way of understanding the essential role of the Hox13 genes in early vertebrate development.This article has an associated 'The people behind the papers' interview.

Restricted expression of cdc25a in the tailbud is essential for formation of the zebrafish posterior body.

The vertebrate body forms from a multipotent stem cell-like progenitor population that progressively contributes newly differentiated cells to the most posterior end of the embryo. How the progenitor population balances proliferation and other cellular functions is unknown due to the difficulty of analyzing cell division in vivo. Here, we show that proliferation is compartmentalized at the posterior end of the embryo during early zebrafish development by the regulated expression of cdc25a, a key controller of mitotic entry. Through the use of a transgenic line that misexpresses cdc25a, we show that this compartmentalization is critical for the formation of the posterior body. Upon misexpression of cdc25a, several essential T-box transcription factors are abnormally expressed, including Spadetail/Tbx16, which specifically prevents the normal onset of myoD transcription, leading to aberrant muscle formation. Our results demonstrate that compartmentalization of proliferation during early embryogenesis is critical for both extension of the vertebrate body and differentiation of the multipotent posterior progenitor cells to the muscle cell fate.

Wnt signaling and tbx16 form a bistable switch to commit bipotential progenitors to mesoderm.

Anterior to posterior growth of the vertebrate body is fueled by a posteriorly located population of bipotential neuro-mesodermal progenitor cells. These progenitors have a limited rate of proliferation and their maintenance is crucial for completion of the anterior-posterior axis. How they leave the progenitor state and commit to differentiation is largely unknown, in part because widespread modulation of factors essential for this process causes organism-wide effects. Using a novel assay, we show that zebrafish Tbx16 (Spadetail) is capable of advancing mesodermal differentiation cell-autonomously. Tbx16 locks cells into the mesodermal state by not only activating downstream mesodermal genes, but also by repressing bipotential progenitor genes, in part through a direct repression of sox2. We demonstrate that tbx16 is activated as cells move from an intermediate Wnt environment to a high Wnt environment, and show that Wnt signaling activates the tbx16 promoter. Importantly, high-level Wnt signaling is able to accelerate mesodermal differentiation cell-autonomously, just as we observe with Tbx16. Finally, because our assay for mesodermal commitment is quantitative we are able to show that the acceleration of mesodermal differentiation is surprisingly incomplete, implicating a potential separation of cell movement and differentiation during this process. Together, our data suggest a model in which high levels of Wnt signaling induce a transition to mesoderm by directly activating tbx16, which in turn acts to irreversibly flip a bistable switch, leading to maintenance of the mesodermal fate and repression of the bipotential progenitor state, even as cells leave the initial high-Wnt environment.

Brachyury establishes the embryonic mesodermal progenitor niche.

Formation of the early vertebrate embryo depends on a Brachyury/Wnt autoregulatory loop within the posterior mesodermal progenitors. We show that exogenous retinoic acid (RA), which dramatically truncates the embryo, represses expression of the zebrafish brachyury ortholog no tail (ntl), causing a failure to sustain the loop. We found that Ntl functions normally to protect the autoregulatory loop from endogenous RA by directly activating cyp26a1 expression. Thus, the embryonic mesodermal progenitors uniquely establish their own niche--with Brachyury being essential for creating a domain of high Wnt and low RA signaling--rather than having a niche created by separate support cells.

A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations.

The ability to visualize and manipulate cell fate and gene expression in specific cell populations has made gene expression systems valuable tools in developmental biology studies. Here, we describe a new system that uses the E. coli tryptophan repressor and its upstream activation sequence (TrpR/tUAS) to drive gene expression in stable zebrafish transgenic lines and in mammalian cells. We show that TrpR/tUAS transgenes are not silenced in subsequent generations of zebrafish, which is a major improvement over some of the existing systems, such as Gal4/gUAS and the Q-system. TrpR transcriptional activity can be tuned by mutations in its DNA-binding domain, or silenced by Gal80 when fused to the Gal4 activation domain. In cases in which more than one cell population needs to be manipulated, TrpR/tUAS can be used in combination with other, existing systems.

A novel cold-sensitive mutant of ntla reveals temporal roles of brachyury in zebrafish.

BACKGROUND: With the exception of the head, the vertebrate embryonic body is formed progressively in an anterior-posterior direction, originating from a posteriorly located bipotential neural-mesodermal progenitor population. The T-box transcription factor Brachyury is expressed within the progenitors and is essential for the formation of the posterior mesoderm. A novel cold-sensitive mutant of Zebrafish Brachyury (ntla(cs) ) is described that allows exploration of the temporal role of this key factor. RESULTS: The ntla(cs) mutant is used to show that Ntla has an essential role during early gastrulation, but as gastrulation proceeds the importance of Ntla declines as Ntlb acquires a capacity to form the posterior mesoderm. Remarkably, ntla(cs) embryos held at the nonpermissive temperature just during the gastrula stages show recovery of normal levels of mesodermal gene expression, demonstrating the plasticity of the posterior progenitors. CONCLUSION: ntla(cs) is a valuable tool for exploring the processes forming the posterior body since it allows temporally specific activation and inactivation of Brachyury function. It is used here to show the changing roles of Ntla during early development and the dynamics of the neuromesodermal progenitors. Developmental Dynamics 245:874-880, 2016. © 2016 Wiley Periodicals, Inc.

Tbx16 and Msgn1 are required to establish directional cell migration of zebrafish mesodermal progenitors.

The epithelial to mesenchymal transition (EMT) is an essential process that occurs repeatedly during embryogenesis whereby stably adherent cells convert to an actively migrating state. While much is known about the factors and events that initiate the EMT, the steps that cells undergo to become directionally migratory are far less well understood. Zebrafish embryos lacking the transcription factors Tbx16/Spadetail and Mesogenin1 (Msgn1) are a valuable system for investigating the EMT. Mesodermal cells in these embryos are unable to perform the EMT necessary to leave the most posterior end of the body (the tailbud) and join the pre-somitic mesoderm, a process that is conserved in all vertebrates. It has previously been very difficult to study this EMT in vertebrates because of the multiple cell types in the tailbud and the morphogenetic changes the whole embryo undergoes. Here, we describe a novel tissue explant system for imaging the mesodermal cell EMT in vivo that allows us to investigate the requirements for cells to acquire migratory properties during the EMT with high spatio-temporal resolution. This method revealed that, despite the inability of tbx16;msgn1-deficient cells to leave the tailbud, actin-based protrusions form surprisingly normally in these cells and they become highly motile. However, tbx16;msgn1-deficient cells have specific cell-autonomous defects in the persistence and anterior direction of migration because the lamellipodia they form are not productive in driving anteriorward migration. Additionally, we show that mesoderm morphogenesis and differentiation are separable and that there is a migratory cue that directs mesodermal cell migration that is independent of Tbx16 and Msgn1. This work defines changes that cells undergo as they complete the EMT and provides new insight into the mechanisms required in vivo for cells to become mesenchymal.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

Graded levels of Pax2a and Pax8 regulate cell differentiation during sensory placode formation.

Pax gene haploinsufficiency causes a variety of congenital defects. Renal-coloboma syndrome, resulting from mutations in Pax2, is characterized by kidney hypoplasia, optic nerve malformation, and hearing loss. Although this underscores the importance of Pax gene dosage in normal development, how differential levels of these transcriptional regulators affect cell differentiation and tissue morphogenesis is still poorly understood. We show that differential levels of zebrafish Pax2a and Pax8 modulate commitment and behavior in cells that eventually contribute to the otic vesicle and epibranchial placodes. Initially, a subset of epibranchial placode precursors lie lateral to otic precursors within a single Pax2a/8-positive domain; these cells subsequently move to segregate into distinct placodes. Using lineage-tracing and ablation analyses, we show that cells in the Pax2a/8+ domain become biased towards certain fates at the beginning of somitogenesis. Experiments involving either Pax2a overexpression or partial, combinatorial Pax2a and Pax8 loss of function reveal that high levels of Pax favor otic differentiation whereas low levels increase cell numbers in epibranchial ganglia. In addition, the Fgf and Wnt signaling pathways control Pax2a expression: Fgf is necessary to induce Pax2a, whereas Wnt instructs the high levels of Pax2a that favor otic differentiation. Our studies reveal the importance of Pax levels during sensory placode formation and provide a mechanism by which these levels are controlled.

Suppression of Alk8-mediated Bmp signaling cell-autonomously induces pancreatic beta-cells in zebrafish.

Bmp signaling has been shown to regulate early aspects of pancreas development, but its role in endocrine, and especially beta-cell, differentiation remains unclear. Taking advantage of the ability in zebrafish embryos to cell-autonomously modulate Bmp signaling in single cells, we examined how Bmp signaling regulates the ability of individual endodermal cells to differentiate into beta-cells. We find that specific temporal windows of Bmp signaling prevent beta-cell differentiation. Thus, future dorsal bud-derived beta-cells are sensitive to Bmp signaling specifically during gastrulation and early somitogenesis stages. In contrast, ventral pancreatic cells, which require an early Bmp signal to form, do not produce beta-cells when exposed to Bmp signaling at 50 hpf, a stage when the ventral bud-derived extrapancreatic duct is the main source of new endocrine cells. Importantly, inhibiting Bmp signaling within endodermal cells via genetic means increased the number of beta-cells, at early and late stages. Moreover, inhibition of Bmp signaling in the late stage embryo using dorsomorphin, a chemical inhibitor of Bmp receptors, significantly increased beta-cell neogenesis near the extrapancreatic duct, demonstrating the feasibility of pharmacological approaches to increase beta-cell numbers. Our in vivo single-cell analyses show that whereas Bmp signaling is necessary initially for formation of the ventral pancreas, differentiating endodermal cells need to be protected from exposure to Bmps during specific stages to permit beta-cell differentiation. These results provide important unique insight into the intercellular signaling environment necessary for in vivo and in vitro generation of beta-cells.

A single-cell transcriptional timelapse of mouse embryonic development, from gastrula to pup.

The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.

Completion of the epithelial to mesenchymal transition in zebrafish mesoderm requires Spadetail.

The process of gastrulation is highly conserved across vertebrates on both the genetic and morphological levels, despite great variety in embryonic shape and speed of development. This mechanism spatially separates the germ layers and establishes the organizational foundation for future development. Mesodermal identity is specified in a superficial layer of cells, the epiblast, where cells maintain an epithelioid morphology. These cells involute to join the deeper hypoblast layer where they adopt a migratory, mesenchymal morphology. Expression of a cascade of related transcription factors orchestrates the parallel genetic transition from primitive to mature mesoderm. Although the early and late stages of this process are increasingly well understood, the transition between them has remained largely mysterious. We present here the first high resolution in vivo observations of the blebby transitional morphology of involuting mesodermal cells in a vertebrate embryo. We further demonstrate that the zebrafish spadetail mutation creates a reversible block in the maturation program, stalling cells in the transition state. This mutation creates an ideal system for dissecting the specific properties of cells undergoing the morphological transition of maturing mesoderm, as we demonstrate with a direct measurement of cell-cell adhesion.

Squinting at the zebrafish axis.

The Nodal family of signaling molecules includes critical intercellular regulators of early vertebrate development. In a recent issue of Nature, maternal transcripts encoding the zebrafish nodal squint were shown to be localized to the future organizer region by the four-cell stage, providing the earliest evidence of embryonic axis asymmetry in the zebrafish embryo.

One-Eyed Pinhead and Spadetail are essential for heart and somite formation.

Mutant analysis in the zebrafish Danio rerio has demonstrated distinct developmental roles for the T-box transcription factor Spadetail (Spt) and the Nodal-receptor cofactor One-Eyed Pinhead (Oep) in the formation of mesoderm and endoderm. Here, we show that spt and oep genetically interact and are together essential for the formation of cardiac and somitic mesoderm. These two mesodermal defects are dependent on different effectors of Nodal signalling; cardiac mesoderm formation involves the mix-like transcription factor Bonnie and Clyde (Bon), whereas somitogenesis is dependent on a different pathway. Analysis of the somite defect in Zoep;spt embryos has provided insights into the control of somitic mesoderm formation by Spt, which was previously implicated in the regulation of cell adhesion and motility. We show that the failure to form somites in Zoep;spt embryos is independent of this and that Spt must have an additional function. We propose that the major role of Spt in somitogenesis is to promote the differentiation of presomitic mesoderm from tailbud progenitors by antagonizing progenitor-type gene expression and behaviour.

Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud.

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.

Developmental biology: micro(RNA)-managing nodal.

Nodal signaling plays an essential role during the induction and patterning of vertebrate embryonic tissue types. In Xenopus and zebrafish, microRNAs regulate Nodal signaling, but surprisingly by very different mechanisms.