Controlling human organoid symmetry breaking reveals signaling gradients drive segmentation clock waves.

Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.

Stepwise emergence of the neuronal gene expression program in early animal evolution.

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.

Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

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.

Changes in genome architecture and transcriptional dynamics progress independently of sensory experience during post-natal brain development.

Both transcription and three-dimensional (3D) architecture of the mammalian genome play critical roles in neurodevelopment and its disorders. However, 3D genome structures of single brain cells have not been solved; little is known about the dynamics of single-cell transcriptome and 3D genome after birth. Here, we generated a transcriptome (3,517 cells) and 3D genome (3,646 cells) atlas of the developing mouse cortex and hippocampus by using our high-resolution multiple annealing and looping-based amplification cycles for digital transcriptomics (MALBAC-DT) and diploid chromatin conformation capture (Dip-C) methods and developing multi-omic analysis pipelines. In adults, 3D genome "structure types" delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first post-natal month. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, we examine allele-specific structure of imprinted genes, revealing local and chromosome (chr)-wide differences. These findings uncover an unknown dimension of neurodevelopment.

Adhesion-Based Self-Organization in Tissue Patterning.

Since the proposal of the differential adhesion hypothesis, scientists have been fascinated by how cell adhesion mediates cellular self-organization to form spatial patterns during development. The search for molecular tool kits with homophilic binding specificity resulted in a diverse repertoire of adhesion molecules. Recent understanding of the dominant role of cortical tension over adhesion binding redirects the focus of differential adhesion studies to the signaling function of adhesion proteins to regulate actomyosin contractility. The broader framework of differential interfacial tension encompasses both adhesion and nonadhesion molecules, sharing the common function of modulating interfacial tension during cell sorting to generate diverse tissue patterns. Robust adhesion-based patterning requires close coordination between morphogen signaling, cell fate decisions, and changes in adhesion. Current advances in bridging theoretical and experimental approaches present exciting opportunities to understand molecular, cellular, and tissue dynamics during adhesion-based tissue patterning across multiple time and length scales.

The Clockwork Embryo: Mechanisms Regulating Developmental Rate.

Organismal development requires the reproducible unfolding of an ordered sequence of discrete steps (cell fate determination, migration, tissue folding, etc.) in both time and space. Here, we review the mechanisms that grant temporal specificity to developmental steps, including molecular clocks and timers. Individual timing mechanisms must be coordinated with each other to maintain the overall developmental sequence. However, phenotypic novelties can also arise through the modification of temporal patterns over the course of evolution. Two main types of variation in temporal patterning characterize interspecies differences in developmental time: allochrony, where the overall developmental sequence is either accelerated or slowed down while maintaining the relative duration of individual steps, and heterochrony, where the duration of specific developmental steps is altered relative to the rest. New advances in in vitro modeling of mammalian development using stem cells have recently enabled the revival of mechanistic studies of allochrony and heterochrony. In both cases, differences in the rate of basic cellular functions such as splicing, translation, protein degradation, and metabolism seem to underlie differences in developmental time. In the coming years, these studies should identify the genetic differences that drive divergence in developmental time between species.

An FGF timer for zygotic genome activation.

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timing of gene induction during the earliest phases of embryogenesis. Here we used high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate Ciona with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by ERK-mediated derepression of the ERF repressor. Depletion of ERF results in ectopic activation of FGF target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the eight- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also used by vertebrates.

Building human artery and vein endothelial cells from pluripotent stem cells, and enduring mysteries surrounding arteriovenous development.

Owing to their manifold roles in health and disease, there have been intense efforts to synthetically generate blood vessels in vitro from human pluripotent stem cells (hPSCs). However, there are multiple types of blood vessel, including arteries and veins, which are molecularly and functionally different. How can we specifically generate either arterial or venous endothelial cells (ECs) from hPSCs in vitro? Here, we summarize how arterial or venous ECs arise during embryonic development. VEGF and NOTCH arbitrate the bifurcation of arterial vs. venous ECs in vivo. While manipulating these two signaling pathways biases hPSC differentiation towards arterial and venous identities, efficiently generating these two subtypes of ECs has remained challenging until recently. Numerous questions remain to be fully addressed. What is the complete identity, timing and combination of extracellular signals that specify arterial vs. venous identities? How do these extracellular signals intersect with fluid flow to modulate arteriovenous fate? What is a unified definition for endothelial progenitors or angioblasts, and when do arterial vs. venous potentials segregate? How can we regulate hPSC-derived arterial and venous ECs in vitro, and generate organ-specific ECs? In turn, answers to these questions could avail the production of arterial and venous ECs from hPSCs, accelerating vascular research, tissue engineering, and regenerative medicine.

Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

Generative models of morphogenesis in developmental biology.

Understanding the mechanism by which cells coordinate their differentiation and migration is critical to our understanding of many fundamental processes such as wound healing, disease progression, and developmental biology. Mathematical models have been an essential tool for testing and developing our understanding, such as models of cells as soft spherical particles, reaction-diffusion systems that couple cell movement to environmental factors, and multi-scale multi-physics simulations that combine bottom-up rule-based models with continuum laws. However, mathematical models can often be loosely related to data or have so many parameters that model behaviour is weakly constrained. Recent methods in machine learning introduce new means by which models can be derived and deployed. In this review, we discuss examples of mathematical models of aspects of developmental biology, such as cell migration, and how these models can be combined with these recent machine learning methods.

Peeking under the hood of early embryogenesis: Using tools and synthetic biology to understand native control systems and sculpt tissues.

Early embryogenesis requires rapid division of pluripotent blastomeres, regulated genome activation, precise spatiotemporal signaling to pattern cell fate, and morphogenesis to shape primitive tissue architectures. The complexity of this process has inspired researchers to move beyond simple genetic perturbation into engineered devices and synthetic biology tools to permit temporal and spatial manipulation of the control systems guiding development. By precise alteration of embryo organization, it is now possible to advance beyond basic analytical strategies and directly test the sufficiency of models for developmental regulation. Separately, advances in micropatterning and embryoid culture have facilitated the bottom-up construction of complex embryo tissues allowing ex vivo systems to recapitulate even later stages of development. Embryos fertilized and grown ex vivo offer an excellent opportunity to exogenously perturb fundamental pathways governing embryogenesis. Here we review the technologies developed to thermally modulate the embryo cell cycle, and optically regulate morphogen and signaling pathways in space and time, specifically in the blastula embryo. Additionally, we highlight recent advances in cell patterning in two and three dimensions that have helped reveal the self-organizing properties and gene regulatory networks guiding early embryo organization.

ABCG2-Expressing Clonal Repopulating Endothelial Cells Serve to Form and Maintain Blood Vessels.

BACKGROUND: Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. METHODS: We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs. RESULTS: In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs. CONCLUSIONS: These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

'Chip'-ing away at morphogenesis - application of organ-on-chip technologies to study tissue morphogenesis.

Emergent cell behaviors that drive tissue morphogenesis are the integrated product of instructions from gene regulatory networks, mechanics and signals from the local tissue microenvironment. How these discrete inputs intersect to coordinate diverse morphogenic events is a critical area of interest. Organ-on-chip technology has revolutionized the ability to construct and manipulate miniaturized human tissues with organotypic three-dimensional architectures in vitro. Applications of organ-on-chip platforms have increasingly transitioned from proof-of-concept tissue engineering to discovery biology, furthering our understanding of molecular and mechanical mechanisms that operate across biological scales to orchestrate tissue morphogenesis. Here, we provide the biological framework to harness organ-on-chip systems to study tissue morphogenesis, and we highlight recent examples where organ-on-chips and associated microphysiological systems have enabled new mechanistic insight in diverse morphogenic settings. We further highlight the use of organ-on-chip platforms as emerging test beds for cell and developmental biology.

Symbiosis of disciplines: how can developmental biologists join conservationists in sustaining and restoring earth's biodiversity?

What can developmental biology contribute toward mitigating the consequences of anthropogenic assaults on the environment and climate change? In this Spotlight article, we advocate a developmental biology that takes seriously Lynn Margulis' claim that 'the environment is part of the body'. We believe this to be a pre-condition for developmental biology playing important roles in conservation and environmental restoration. We need to forge a developmental biology of the holobiont - the multi-genomic physiologically integrated organism that is also a functional biome. To this end, we highlight how developmental biology needs to explore more deeply the interactions between developing organisms, and their chemical, physical and biotic environments.

Osteoblasts pattern endothelium and somatosensory axons during zebrafish caudal fin organogenesis.

Skeletal elements frequently associate with vasculature and somatosensory nerves, which regulate bone development and homeostasis. However, the deep, internal location of bones in many vertebrates has limited in vivo exploration of the neurovascular-bone relationship. Here, we use the zebrafish caudal fin, an optically accessible organ formed of repeating bony ray skeletal units, to determine the cellular relationship between nerves, bones and endothelium. In adult zebrafish, we establish the presence of somatosensory axons running through the inside of the bony fin rays, juxtaposed with osteoblasts on the inner hemiray surface. During development we show that the caudal fin progresses through sequential stages of endothelial plexus formation, bony ray addition, ray innervation and endothelial remodeling. Surprisingly, the initial stages of fin morphogenesis proceed normally in animals lacking either fin endothelium or somatosensory nerves. Instead, we find that sp7+ osteoblasts are required for endothelial remodeling and somatosensory axon innervation in the developing fin. Overall, this study demonstrates that the proximal neurovascular-bone relationship in the adult caudal fin is established during fin organogenesis and suggests that ray-associated osteoblasts pattern axons and endothelium.

The fetal lineage is susceptible to Zika virus infection within days of fertilization.

Adults contracting Zika virus (ZIKV) typically exhibit mild symptoms, yet ZIKV infection of pregnant individuals can cause miscarriage or birth defects in their offspring. Many studies have focused on maternal-to-fetal ZIKV transmission via blood and placenta. Notably, however, ZIKV is also transmitted sexually, raising the possibility that ZIKV could infect the embryo shortly after fertilization, long before the placenta is established. Here, we evaluate the consequences of ZIKV infection in mouse embryos during the first few days of embryogenesis. We show that divergent strains of ZIKV can infect the fetal lineage and can cause developmental arrest, raising concern for the developmental consequences of sexual ZIKV transmission. This article has an associated 'The people behind the papers' interview.

Fly Me to the Micron: Microtechnologies for Drosophila Research.

Multicellular model organisms, such as Drosophila melanogaster (fruit fly), are frequently used in a myriad of biological research studies due to their biological significance and global standardization. However, traditional tools used in these studies generally require manual handling, subjective phenotyping, and bulk treatment of the organisms, resulting in laborious experimental protocols with limited accuracy. Advancements in microtechnology over the course of the last two decades have allowed researchers to develop automated, high-throughput, and multifunctional experimental tools that enable novel experimental paradigms that would not be possible otherwise. We discuss recent advances in microtechnological systems developed for small model organisms using D. melanogaster as an example. We critically analyze the state of the field by comparing the systems produced for different applications. Additionally, we suggest design guidelines, operational tips, and new research directions based on the technical and knowledge gaps in the literature. This review aims to foster interdisciplinary work by helping engineers to familiarize themselves with model organisms while presenting the most recent advances in microengineering strategies to biologists.