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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 indexing3to 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 data4–8from 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.

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.

Motor neurons in the central nervous system often lie in a continuous topographic map, where neurons that innervate different body parts are spatially intermingled. This is the case for the efferent neurons of the vagus nerve, which innervate diverse muscle and organ targets in the head and viscera for brain-body communication. It remains elusive how neighboring motor neurons with different fixed peripheral axon targets develop the separate somatodendritic (input) connectivity they need to generate spatially precise body control. Here, we show that vagus motor neurons in the zebrafish indeed generate spatially appropriate peripheral responses to focal sensory stimulation even when they are transplanted into ectopic positions within the topographic map, indicating that circuit refinement occurs after the establishment of coarse topography. Refinement depends on motor neuron synaptic transmission, suggesting that an experience-dependent periphery-to-brain feedback mechanism establishes specific input connectivity among intermingled motor populations.

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of ~150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type’s specification, as well as ‘cell-type homologs’ across vertebrate evolution.

The mechanisms that allow cells to bypass anti–vascular endothelial growth factor A (VEGFA) therapy remain poorly understood. Here we use zebrafish to investigate this question and first show that vegfaa mutants display a severe vascular phenotype that can surprisingly be rescued to viability by vegfaa messenger RNA injections at the 1-cell stage. Using vegfaa mutants as an in vivo test tube, we found that zebrafish Vegfbb, Vegfd, and Pgfb can also rescue these animals to viability. Taking advantage of a new vegfr1 tyrosine kinase–deficient mutant, we determined that Pgfb rescues vegfaa mutants via Vegfr1. Altogether, these data reveal potential resistance routes against current anti-VEGFA therapies. In order to circumvent this resistance, we engineered and validated new dominant negative Vegfa molecules that by trapping Vegf family members can block vascular development. Thus, our results show that Vegfbb, Vegfd, and Pgfb can sustain vascular development in the absence of VegfA, and our newly engineered Vegf molecules expand the toolbox for basic research and antiangiogenic therapy.

The mechanisms that allow cells to bypass anti–vascular endothelial growth factor A (VEGFA) therapy remain poorly understood. Here we use zebrafish to investigate this question and first show that vegfaamutants display a severe vascular phenotype that can surprisingly be rescued to viability by vegfaamessenger RNA injections at the 1-cell stage. Using vegfaamutants as an in vivo test tube, we found that zebrafish Vegfbb, Vegfd, and Pgfb can also rescue these animals to viability. Taking advantage of a new vegfr1tyrosine kinase–deficient mutant, we determined that Pgfb rescues vegfaamutants via Vegfr1. Altogether, these data reveal potential resistance routes against current anti-VEGFA therapies. In order to circumvent this resistance, we engineered and validated new dominant negative Vegfa molecules that by trapping Vegf family members can block vascular development. Thus, our results show that Vegfbb, Vegfd, and Pgfb can sustain vascular development in the absence of VegfA, and our newly engineered Vegf molecules expand the toolbox for basic research and antiangiogenic therapy.

TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.

Transcript elongation is a critical step in the production of mature messenger RNAs. Many factors have been identified that are required for transcript elongation, including Spt5. Studies in yeast determined that spt5is required for cell viability, and analyses in Drosophilaindicate Spt5 is localized to sites of active transcription, suggesting it is required generally for transcription. However, the requirement for spt5for cell viability in a metazoan organism has not been addressed. We determined that zebrafish foggy/spt5is required cell‐autonomously for the posterior migration of facial branchiomotor neurons from rhombomere 4 (r4) into r6 and r7 of the hindbrain. These genetic mosaics also give us the unique opportunity to determine whether spt5is required for mRNA transcription equivalently at all loci by addressing two processes within the same cell—neuronal migration and cell viability. In a wild‐type host, spt5null facial branchiomotor neurons survive to at least 5 days postfertilization while failing to migrate posteriorly. This finding indicates that spt5‐dependent transcript elongation is required cell‐autonomously for a complex cell migration but not for the survival of these same cells. This work provides evidence that transcript elongation is not a global mechanism equivalently required by all loci and may actually be under more strict developmental regulation. Developmental Dynamics 234:651–658, 2005. © 2005 Wiley‐Liss, Inc.

The hindbrain is responsible for controlling essential functions such as respiration and heart beat that we literally do not think about most of the time. In addition, cranial nerves projecting from the hindbrain control muscles in the jaw, eye, and face, and receive sensory input from these same areas. In all vertebrates that have been studied, the hindbrain passes through a segmented phase shortly after the neural tube has formed, with a series of seven bulges—the rhombomeres—forming along the anterior-posterior extent of the neural tube. Our current understanding of vertebrate hindbrain development comes from integrating data from several model systems. Work on the chick has helped us to understand the cell biology of the rhombomeres, whereas the power of mouse molecular genetics has allowed investigation of the molecular mechanisms underlying their development. This review focuses on the special insights that the zebrafish system has provided to our understanding of hindbrain development. As we will discuss, work in the zebrafish has elucidated inductive events that specify the presumptive hindbrain domain and has identified genes required for hindbrain segmentation and the specification of segment identities. © 2002 Wiley-Liss, Inc.

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr– phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

In a genetic screen, we isolated a mutation that perturbed motor axon outgrowth, neurogenesis, and somitogenesis. Complementation tests revealed that this mutation is an allele of deadly seven(des). By creating genetic mosaics, we demonstrate that the motor axon defect is non-cell autonomous. In addition, we show that the pattern of migration for some neural crest cell populations is aberrant and crest-derived dorsal root ganglion neurons are misplaced. Furthermore, our analysis reveals that desmutant embryos exhibit a neurogenic phenotype. We find an increase in the number of primary motoneurons and in the number of three hindbrain reticulospinal neurons: Mauthner cells, RoL2 cells, and MiD3cm cells. We also find that the number of Rohon–Beard sensory neurons is decreased whereas neural crest-derived dorsal root ganglion neurons are increased in number supporting a previous hypothesis that Rohon–Beard neurons and neural crest form an equivalence group during development. Mutations in genes involved in Notch–Delta signaling result in defects in somitogenesis and neurogenesis. We found that overexpressing an activated form of Notchdecreased the number of Mauthner cells in desmutants indicating that desfunctions via the Notch–Delta signaling pathway to control the production of specific cell types within the central and peripheral nervous systems.

Forward genetic analyses can reveal important developmental regulatory genes and how they function to pattern morphology. This is because a mutated gene can produce a novel, sometimes beautiful, phenotype that, like the normal phenotype, immediately seems worth understanding. Generally the loss-of-function mutant phenotype is simplified from the wild-type one, and often the nature of the pattern simplification allows one to deduce how the wild-type gene contributes to patterning the normal, more complex, morphology. This truism seems no less valid for the vertebrate head skeleton than for other and simpler cases of patterning in multicellular plants and animals. To show this, we review selected zebrafish craniofacial mutants. “Midline group” mutations, in genes functioning in one of at least three signal transduction pathways, lead to neurocranial pattern truncations that are primarily along the mediolateral axis. Mutation of lazarus/pbx4,encoding a hoxgene partner, and mutation of valentino/kreisler,a hoxgene regulator, produce anterior–posterior axis disruptions of pharyngeal cartilages. Dorsoventral axis patterning of the same cartilages is disrupted in sucker/endothelin-1mutants. We infer that different signal transduction pathways pattern cartilage development along these three separate axes. Patterning of at least the anterior–posterior and dorsoventral axes have been broadly conserved, e.g., reduced Endothelin-1 signaling similarly perturbs cartilage specification in chick, mouse, and zebrafish. We hypothesize that Endothelin-1 also is an upstream organizer of the patterns of cellular interactions during cartilage morphogenesis.

Rhombomeres are segmental units of the developing vertebrate hindbrain that underlie the reiterated organisation of cranial neural crest migration and neuronal differentiation. valentino (val), a zebrafish homologue of the mouse bzip transcription factor-encoding gene, kreisler, is required for segment boundary formation caudal to rhombomere 4 (r4). val is normally expressed in r5/6 and is required for cells to contribute to this region. In val− mutants, rX, a region one rhombomere in length and of mixed identity, lies between r4 and r7. While a number of genes involved in establishing rhombomeric identity are known, it is still largely unclear how segmental integrity is established and boundaries are formed. Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are candidates for functioning in rhombomere boundary formation. Indeed, expression of the receptor ephB4a coincides with val in r5/6, whilst ephrin-B2a, which encodes a ligand for EphB4a, is expressed in r4 and r7, complementary to the domain of val expression. Here we show that in val− embryos, ephB4a expression is downregulated and ephrin-B2a expression is upregulated between r4 and r7, indicating that Val is normally required to establish the mutually exclusive expression domains of these two genes. We show that juxtaposition of ephB4a-expressing cells and ephrin-B2a-expressing cells in the hindbrain leads to boundary formation. Loss of the normal spatial regulation of eph/ephrin expression in val mutants correlates not only with absence of boundaries but also with the inability of mutant cells to contribute to wild-type r5/6. Using a genetic mosaic approach, we show that spatially inappropriate Eph signalling underlies the repulsion of val− cells from r5/6. We propose that Val controls eph expression and that interactions between EphB4a and Ephrin-B2a mediate cell sorting and boundary formation in the segmenting caudal hindbrain.

Two types of mutant allele, one leaky and one null, have been generated by gene targeting at the N-myc locus in embryonic stem cells and the phenotypes of mice homozygous for these mutations have been described. These mutations have shown that N-myc has a number of functions during development, including a role in branching morphogenesis in the lung, which manifests itself at birth in mice homozygous for the leaky allele, and roles in the development of the mesonephric tubules, the neuroepithelium, the sensory ganglia, the gut and the heart, which become evident at midgestation in embryos homozygous for the null allele. In an attempt to define roles for N-myc at other stages of development, we have combined the two types of N-myc mutant allele in a compound heterozygote that as a result contains approximately 15% of normal levels of N-Myc protein. Compound heterozygotes died during gestation at a time intermediate to the times of death of embryos homozygous for either mutation individually, and their death appeared to result from cardiac failure stemming from hypoplasia of the compact subepicardial layer of the myocardium. Investigation of the expression pattern of N-myc and various markers of differentiation in wild-type and compound heterozygote mutant hearts has suggested that N-myc may function in maintaining the proliferation and/or preventing the differentiation of compact layer myocytes. This study illustrates the importance of generating different mutations at a given locus to elucidate fully the function of a particular gene during development.

Mutational analysis can serve both to identify new genes essential for patterning embryonic development and to determine their functions. Here we describe the identifica-tion and phenotypic characterization of alleles of valentino, which we recovered in a genetic screen that sought to identify mutations in the zebrafish that disrupt region-specific gene expression patterns in the embryonic brain. valentino is required for normal hindbrain segmentation and the hindbrain of valentino mutant embryos is shortened by the length of one rhombomere. We demon-strate that valentino is required cell-autonomously in the development of rhombomeres 5 and 6, and propose that valentino functions in the subdivision and expansion of a common precursor region in the presumptive hindbrain into the definitive rhombomeres 5 and 6. These results provide genetic evidence for a two-segment periodicity in the hindbrain and suggest that this periodicity arises sequentially, through the specification and later subdivi-sion of a two-rhombomere unit, or ‘protosegment’.

The developing hindbrain is organized into a series of segments termed rhombomeres which represent lineage restricted compartments correlating with domains of gene expression and neuronal differentiation. In this study, we investigate the processes of hindbrain segmentation and the acquisition of segmental identity by analyzing the expression of zebrafish hox genes in the hindbrains of normal fish and fish with a loss-of-function mutation in the segmentation gene valentino (val, the homologue of mouse kreisler; Moens, C. B., Cordes, S. P. Giorgianni, M. W., Barsh, G. S. and Kimmel, C. B. (1998). Development 125, 381-391). We find that zebrafish hox genes generally have similar expression profiles to their murine and avian counterparts, although there are several differences in timing and spatial extent of expression which may underlie some of the functional changes that have occurred along the separate evolutionary lineages of teleosts and tetrapods. Our analysis of hox gene expression in val− embryos confirms that the val gene product is important for subdivision of the presumptive rhombomere 5 and 6 territory into definitive rhom-bomeres, suggests that the val gene product plays a critical role in regulating hox gene transcription, and indicates that some neural crest cells are inappropriately specified in val− embryos. Our analysis of gene expression at several developmental stages has allowed us to infer dif-ferences between primary and secondary defects in the val mutant: we find that extended domains of expression for some hox genes are secondary, late phenomena potentially resulting from inappropriate cell mixing or lack of normal inter-rhombomeric interactions in the caudal hindbrain.

The mechanisms underlying neuronal specification and axonogenesis in the vertebrate hindbrain are poorly under-stood. To address these questions, we have employed anatomical methods and mutational analysis to character-ize the branchiomotor neurons in the zebrafish embryo. The zebrafish branchiomotor system is similar to those in the chick and mouse, except for the location of the nVII and nIX branchiomotor neurons. Developmental analyses of genes expressed by branchiomotor neurons suggest that the different location of the nVII neurons in the zebrafish may result from cell migration. To gain insight into the mechanisms underlying the organization and axonogenesis of these neurons, we examined the development of the branchiomotor pathways in neuronal mutants. The valentinob337 mutation blocks the formation of rhombomeres 5 and 6, and severely affects the development of the nVII and nIX motor nuclei. The cyclopsb16 mutation deletes ventral midline cells in the neural tube, and leads to a severe disruption of most bran-chiomotor nuclei and axon pathways. These results demon-strate that rhombomere-specific cues and ventral midline cells play important roles in the development of the bran-chiomotor pathways.