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.

Dissecting cell identity via network inference and in silico gene perturbation.

Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks1. Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms-mouse and human haematopoiesis, and zebrafish embryogenesis-and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto, an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a. Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation.

A squash and a squeeze.

Advanced imaging techniques reveal details of the interactions between the two layers of the embryonic midgut that influence its ultimate shape.

Reciprocal regulation of endothelial-mesenchymal transition by MAPK7 and EZH2 in intimal hyperplasia and coronary artery disease.

Endothelial-mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFß1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.

Mesenchymal proteases and tissue fluidity remodel the extracellular matrix during airway epithelial branching in the embryonic avian lung.

Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we have found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase 2, which is necessary for branch extension but for not branch initiation. As branches extend, tenascin C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

Long-Range Signaling Activation and Local Inhibition Separate the Mesoderm and Endoderm Lineages.

Specification of the three germ layers by graded Nodal signaling has long been seen as a paradigm for patterning through a single morphogen gradient. However, by exploiting the unique properties of the zebrafish embryo to capture the dynamics of signaling and cell fate allocation, we now demonstrate that Nodal functions in an incoherent feedforward loop, together with Fgf, to determine the pattern of endoderm and mesoderm specification. We show that Nodal induces long-range Fgf signaling while simultaneously inducing the cell-autonomous Fgf signaling inhibitor Dusp4 within the first two cell tiers from the margin. The consequent attenuation of Fgf signaling in these cells allows specification of endoderm progenitors, while the cells further from the margin, which receive Nodal and/or Fgf signaling, are specified as mesoderm. This elegant model demonstrates the necessity of feedforward and feedback interactions between multiple signaling pathways for providing cells with temporal and positional information.

Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.

Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Ectoderm-mesoderm crosstalk in the embryonic limb: The role of fibroblast growth factor signaling.

In this commentary we focus on the function of FGFs during limb development and morphogenesis. Our goal is to understand, interpret and, when possible, reconcile the interesting findings and conflicting results that remain unexplained. For example, the cell death pattern observed after surgical removal of the AER versus genetic removal of the AER-Fgfs is strikingly different and the field is at an impasse with regard to an explanation. We also discuss the idea that AER function may involve signaling components in addition to the AER-FGFs and that signaling from the non-AER ectoderm may also have a significant contribution. We hope that a re-evaluation of current studies and a discussion of outstanding questions will motivate new experiments, especially considering the availability of new technologies, that will fuel further progress toward understanding the intricate ectoderm-to-mesoderm crosstalk during limb development. Developmental Dynamics 246:208-216, 2017. © 2016 Wiley Periodicals, Inc.

Tamoxifen-dependent, inducible Bmp2CreER drives selective recombinase activity in early interdigital mesenchyme and digit collateral ligaments.

During limb development, the interdigital mesenchyme has been proposed to play a signaling role instructing morphogenesis of different digit types, as well as undergoing programmed cell death necessary to free digits in animals not adapted for swimming or flying. We have generated a conditional, tamoxifen-dependent Cre line, Bmp2CreER, which drives highly selective recombination restricted to the distal limb mesoderm, largely restricted to the interdigits, and selectively active in digit ligament but not tendon progenitors at later stages. The Bmp2CreER provides a valuable new tool to dissect roles of interdigital mesenchyme and potentially investigate divergence of ligament and tendon lineages.

Altered retinoic acid signalling underpins dentition evolution.

Small variations in signalling pathways have been linked to phenotypic diversity and speciation. In vertebrates, teeth represent a reservoir of adaptive morphological structures that are prone to evolutionary change. Cyprinid fish display an impressive diversity in tooth number, but the signals that generate such diversity are unknown. Here, we show that retinoic acid (RA) availability influences tooth number size in Cyprinids. Heterozygous adult zebrafish heterozygous for the cyp26b1 mutant that encodes an enzyme able to degrade RA possess an extra tooth in the ventral row. Expression analysis of pharyngeal mesenchyme markers such as dlx2a and lhx6 shows lateral, anterior and dorsal expansion of these markers in RA-treated embryos, whereas the expression of the dental epithelium markers dlx2b and dlx3b is unchanged. Our analysis suggests that changes in RA signalling play an important role in the diversification of teeth in Cyprinids. Our work illustrates that through subtle changes in the expression of rate-limiting enzymes, the RA pathway is an active player of tooth evolution in fish.

Discoidin domain receptors: a proteomic portrait.

The discoidin domain receptors (DDRs) are collagen-binding receptor tyrosine kinases that have been implicated in a number of fundamental biological processes ranging from growth and development to immunoregulation. In this review, we examine how recent proteomic technologies have enriched our understanding of DDR signaling mechanisms. We provide an overview on the use of large-scale proteomic profiling and chemical proteomics to reveal novel insights into DDR therapeutics, signaling networks, and receptor crosstalk. A perspective of how proteomics may be harnessed to answer outstanding fundamental questions including the dynamic regulation of receptor activation kinetics is presented. Collectively, these studies present an emerging molecular portrait of these unique receptors and their functional role in health and disease.

Cyp26 enzymes are required to balance the cardiac and vascular lineages within the anterior lateral plate mesoderm.

Normal heart development requires appropriate levels of retinoic acid (RA) signaling. RA levels in embryos are dampened by Cyp26 enzymes, which metabolize RA into easily degraded derivatives. Loss of Cyp26 function in humans is associated with numerous developmental syndromes that include cardiovascular defects. Although previous studies have shown that Cyp26-deficient vertebrate models also have cardiovascular defects, the mechanisms underlying these defects are not understood. Here, we found that in zebrafish, two Cyp26 enzymes, Cyp26a1 and Cyp26c1, are expressed in the anterior lateral plate mesoderm (ALPM) and predominantly overlap with vascular progenitors (VPs). Although singular knockdown of Cyp26a1 or Cyp26c1 does not overtly affect cardiovascular development, double Cyp26a1 and Cyp26c1 (referred to here as Cyp26)-deficient embryos have increased atrial cells and reduced cranial vasculature cells. Examining the ALPM using lineage tracing indicated that in Cyp26-deficient embryos the myocardial progenitor field contains excess atrial progenitors and is shifted anteriorly into a region that normally solely gives rise to VPs. Although Cyp26 expression partially overlaps with VPs in the ALPM, we found that Cyp26 enzymes largely act cell non-autonomously to promote appropriate cardiovascular development. Our results suggest that localized expression of Cyp26 enzymes cell non-autonomously defines the boundaries between the cardiac and VP fields within the ALPM through regulating RA levels, which ensures a proper balance of myocardial and endothelial lineages. Our study provides novel insight into the earliest consequences of Cyp26 deficiency that underlie cardiovascular malformations in vertebrate embryos.

Calcifying Cystic Odontogenic Tumour: immunohistochemical expression of matrix metalloproteinases, their inhibitors (TIMPs and RECK) and inducer (EMMPRIN).

BACKGROUND: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT. MATERIALS AND METHODS: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments. RESULTS: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003). CONCLUSIONS: We concluded that these proteins/enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour.

Dicer function is required in the metanephric mesenchyme for early kidney development.

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that act as posttranscriptional repressors by binding to the 3'-untranslated region (3'-UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at midgestation (Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA. J Am Soc Nephrol 22: 1053-1063, 2011). To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1, and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the proapoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis and indicate a crucial role for miRNAs in regulating the survival of this lineage.

Sex-specific expression and localization of aromatase and its regulators during embryonic and larval development of Atlantic salmon.

The products of dax1, foxl2a and mis have each been shown to have proliferative and/or differentiative activities during mammalian organogenesis. These factors also play a role in regulating the biosynthesis of estrogen, particularly by modulating the activity of aromatase cyp19a. We demonstrate the transcription and translation of these genes during salmon embryogenesis. We were able to track sex-specific differences in these processes through accurate determination of the sex of each embryo and larva examined from genotyped microsatellites. We detected sex- and stage-specific immunolabeling of the embryonic gut, kidney, gonads, neural cord and skeletal muscle by DAX-1, FOXL2A and MIS. These results indicate the potential of these factors to mediate proliferation and/or differentiation programs during development of these tissues. As well, immunolabeling of skeletal muscle by CYP19B1 throughout the study reveals probable neurogenic activity associated with peripheral radial glial cells and the growing embryonic musculature.

Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix.

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process.

Araf kinase antagonizes Nodal-Smad2 activity in mesendoderm development by directly phosphorylating the Smad2 linker region.

Smad2/3-mediated transforming growth factor ß signalling and the Ras-Raf-Mek-Erk cascade have important roles in stem cell and development and tissue homeostasis. However, it remains unknown whether Raf kinases directly crosstalk with Smad2/3 signalling and how this would regulate embryonic development. Here we show that Araf antagonizes mesendoderm induction and patterning activity of Nodal/Smad2 signals in vertebrate embryos by directly inhibiting Smad2 signalling. Knockdown of araf in zebrafish embryos leads to an increase of activated Smad2 with a decrease in linker phosphorylation; consequently, the embryos have excess mesendoderm precursors and are dorsalized. Mechanistically, Araf physically binds to and phosphorylates Smad2 in the linker region with S253 being indispensable in a Mek/Erk-independent manner, thereby attenuating Smad2 signalling by accelerating degradation of activated Smad2. Our findings open avenues for investigating the potential significance of Raf regulation of transforming growth factor ß signalling in versatile biological and pathological processes in the future.

The secreted AdamTS-A metalloprotease is required for collective cell migration.

Members of the ADAMTS family of secreted metalloproteases play crucial roles in modulating the extracellular matrix (ECM) in development and disease. Here, we show that ADAMTS-A, the Drosophila ortholog of human ADAMTS 9 and ADAMTS 20, and of C. elegans GON-1, is required for cell migration during embryogenesis. AdamTS-A is expressed in multiple migratory cell types, including hemocytes, caudal visceral mesoderm (CVM), the visceral branch of the trachea (VBs) and the secretory portion of the salivary gland (SG). Loss of AdamTS-A causes defects in germ cell, CVM and VB migration and, depending on the tissue, AdamTS-A functions both autonomously and non-autonomously. In the highly polarized collective of the SG epithelium, loss of AdamTS-A causes apical surface irregularities and cell elongation defects. We provide evidence that ADAMTS-A is secreted into the SG lumen where it functions to release cells from the apical ECM, consistent with the defects observed in AdamTS-A mutant SGs. We show that loss of the apically localized protocadherin Cad99C rescues the SG defects, suggesting that Cad99C serves as a link between the SG apical membrane and the secreted apical ECM component(s) cleaved by ADAMTS-A. Our analysis of AdamTS-A function in the SG suggests a novel role for ADAMTS proteins in detaching cells from the apical ECM, facilitating tube elongation during collective cell migration.

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy.

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.