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The famous Arrhenius equation is well suited to describing the temperature dependence of chemical reactions but has also been used for complicated biological processes. Here, we evaluate how well the simple Arrhenius equation predicts complex multi‐step biological processes, using frog and fruit fly embryogenesis as two canonical models. We find that the Arrhenius equation provides a good approximation for the temperature dependence of embryogenesis, even though individual developmental intervals scale differently with temperature. At low and high temperatures, however, we observed significant departures from idealized Arrhenius Law behavior. When we model multi‐step reactions of idealized chemical networks, we are unable to generate comparable deviations from linearity. In contrast, we find the two enzymes GAPDH and β‐galactosidase show non‐linearity in the Arrhenius plot similar to our observations of embryonic development. Thus, we find that complex embryonic development can be well approximated by the simple Arrhenius equation regardless of non‐uniform developmental scaling and propose that the observed departure from this law likely results more from non‐idealized individual steps rather than from the complexity of the system. SYNOPSIS Time‐lapse analysis of frog and fruit fly embryogenesis combined with mathematical modeling is performed to examine how well the simple Arrhenius equation predicts complex multi‐step biological processes. The simple Arrhenius equation approximates the temperature dependence of complex embryonic development well. Developmental progression of different stages increases non‐uniformly with temperature. At extreme temperatures, the observed developmental rates are slower than that predicted by the Arrhenius equation. Non‐idealized temperature dependence of embryonic development could be predominantly due to the non‐idealized behavior of individual steps rather than the complexity of the system. Graphical Abstract Time‐lapse analysis of frog and fruit fly embryogenesis combined with mathematical modeling is performed to examine how well the simple Arrhenius equation predicts complex multi‐step biological processes.

High‐throughput ‐omics techniques have revolutionised biology, allowing for thorough and unbiased characterisation of the molecular states of biological systems. However, cellular decision‐making is inherently a unicellular process to which “bulk” ‐omics techniques are poorly suited, as they capture ensemble averages of cell states. Recently developed single‐cell methods bridge this gap, allowing high‐throughput molecular surveys of individual cells. In this review, we cover core concepts of analysis of single‐cell gene expression data and highlight areas of developmental biology where single‐cell techniques have made important contributions. These include understanding of cell‐to‐cell heterogeneity, the tracing of differentiation pathways, quantification of gene expression from specific alleles, and the future directions of cell lineage tracing and spatial gene expression analysis. Graphical Abstract Single‐cell genomic techniques have advanced our understanding of several developmental processes. This Review summarises advances related to generating and analyzing single‐cell transcriptome data and discusses areas of developmental biology that benefited from such technologies.

Mammalian chromosomes fold into arrays of megabase‐sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher‐order organization remains elusive. Here, we investigate TAD higher‐order interactions with Hi‐C through neuronal differentiation and show that they form a hierarchy of domains‐within‐domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree‐like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency. Synopsis Genome‐wide mapping of chromatin architecture reveals a hierarchical folding of chromatin that involves higher‐order domains interactions across the whole chromosomes, reflects epigenomic features and reorganizes upon differentiation‐induced gene expression changes. Chromatin architecture is mapped genome‐wide using Hi‐C and a neuronal differentiation model from mESC to post‐mitotic neurons. Mammalian chromosomes fold hierarchically in a manner that reflects epigenomic features and involves higher‐order domains (metaTADs) up to the chromosome scale. metaTAD topologies are relatively conserved through differentiation, and their reorganization is related to gene expression changes. Polymer modelling shows that hierarchical chromatin folding promotes efficient packaging without the loss of contact specificity. Graphical Abstract Genome‐wide mapping of chromatin architecture reveals a hierarchical folding of chromatin that involves higher‐order domains interactions across the whole chromosomes, reflects epigenomic features and reorganizes upon differentiation‐induced gene expression changes.

Histological analysis of biological tissues by mechanical sectioning is significantly time‐consuming and error‐prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue‐clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands‐on implementation. Graphical Abstract Tissue‐clearing methods have allowed imaging intact biological specimens and understanding biology at a whole‐organism and systems‐level. This Review provides a guide for scientists who would like to setup a clearing protocol, with an emphasis on DISCO methods.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt‐agonistic R‐spondins (RSPOs). Intestinal, stomach and liver Lgr5 + stem cells grow in 3D cultures to form ever‐expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro , duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1‐based cultures, and develop into budding cyst‐like structures (organoids) that expand five‐fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi‐potentiality. The establishment of conditions for long‐term culture and expansion of adult, bi‐potent pancreas progenitors may facilitate novel and tailored therapeutic approaches.

Gene expression oscillators can structure biological events temporally and spatially. Different biological functions benefit from distinct oscillator properties. Thus, finite developmental processes rely on oscillators that start and stop at specific times, a poorly understood behavior. Here, we have characterized a massive gene expression oscillator comprising > 3,700 genes in Caenorhabditis elegans larvae. We report that oscillations initiate in embryos, arrest transiently after hatching and in response to perturbation, and cease in adults. Experimental observation of the transitions between oscillatory and non‐oscillatory states at high temporal resolution reveals an oscillator operating near a Saddle Node on Invariant Cycle (SNIC) bifurcation. These findings constrain the architecture and mathematical models that can represent this oscillator. They also reveal that oscillator arrests occur reproducibly in a specific phase. Since we find oscillations to be coupled to developmental processes, including molting, this characteristic of SNIC bifurcations endows the oscillator with the potential to halt larval development at defined intervals, and thereby execute a developmental checkpoint function. Synopsis The authors investigate a putative developmental clock in C. elegans . Population‐ and single animal‐based analyses uncover a gene expression oscillator that may support a developmental checkpoint function. Extensive rhythmic gene expression in C. elegans larvae is initiated in embryos and is coupled to molting. The oscillator is arrested in a specific phase (normally observed at molt exit) in adults, early L1 and dauer larvae. A bifurcation of the oscillator constitutes a putative developmental checkpoint mechanism. Characteristics of oscillation onset and offset constrain potential oscillator mechanisms as well as mathematical models and their parameters. Graphical Abstract The authors investigate a putative developmental clock in C. elegans . Population‐ and single animal‐based analyses uncover a gene expression oscillator that may support a developmental checkpoint function.

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape. In addition to the Daschous‐Fat‐Dachs planar cell polarity pathway, cell divisions in the developing Drosophila wing are oriented by changes in cell shape caused by circumferential mechanical forces that arise from local differences in proliferation rates.

The function of metabolic state in stemness is poorly understood. Mouse embryonicstem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent statesrepresenting the inner cell mass (ICM) and epiblast embryos. Human embryonic stemcells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic differencebetween these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalentin their energy production, dynamically switching from glycolysis to mitochondrialrespiration on demand. Despite having a more developed and expanding mitochondrialcontent, EpiSC/hESC have low mitochondrial respiratory capacity due to lowcytochrome c oxidase (COX) expression. Similarly, in vivo epiblastssuppress COX levels. These data reveal EpiSC/hESC functional similarity to theglycolytic phenotype in cancer (Warburg effect). We further show thathypoxia‐inducible factor 1α (HIF1α) is sufficient to drive ESC to aglycolytic Activin/Nodal‐dependent EpiSC‐like stage. This metabolic switch duringearly stem‐cell development may be deterministic. Metabolic analyses on naive ESCs versus primed EpiSCs reveal a functional switch fromoxidative phosphorylation to glycolysis. Differential gene expression definesHIF1α and reduced complex IV activity as crucial metabolic regulators duringstem‐cell differentiation.

The Hippo signalling pathway has emerged as a key regulator of organ size, tissue homeostasis, and patterning. Recent studies have shown that two effectors in this pathway, YAP/TAZ, modulate Wnt/β‐catenin signalling through their interaction with β‐catenin or Dishevelled, depending on biological contexts. Here, we identify a novel mechanism through which Hippo signalling inhibits Wnt/β‐catenin signalling. We show that YAP and TAZ, the transcriptional co‐activators in the Hippo pathway, suppress Wnt signalling without suppressing the stability of β‐catenin but through preventing its nuclear translocation. Our results show that YAP/TAZ binds to β‐catenin, thereby suppressing Wnt‐target gene expression, and that the Hippo pathway‐stimulated phosphorylation of YAP, which induces cytoplasmic translocation of YAP, is required for the YAP‐mediated inhibition of Wnt/β‐catenin signalling. We also find that downregulation of Hippo signalling correlates with upregulation of β‐catenin signalling in colorectal cancers. Remarkably, our analysis demonstrates that phosphorylated YAP suppresses nuclear translocation of β‐catenin by directly binding to it in the cytoplasm. These results provide a novel mechanism, in which Hippo signalling antagonizes Wnt signalling by regulating nuclear translocation of β‐catenin. The Hippo pathway effector YAZ is found to bind β‐catenin and prevents its nuclear translocation. The resulting downregulation of Wnt signal transduction provides a new example for intersection of Hippo and Wnt signalling, two key regulatory pathways in animal development

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent ‘+4’ cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5 + CBC cells. Transcriptional profiling of FACS‐sorted Lgr5 + stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell‐enriched genes was defined. Spatial expression patterns were further characterized by mRNA in‐situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5 + stem cells. Lineage tracing using a newly established knock‐in mouse for one of the signature genes, Smoc2 , confirmed its stem cell specificity. Using this resource, we find—and confirm by independent approaches—that the proposed quiescent/‘+4’ stem cell markers Bmi1 , Tert , Hopx and Lrig1 are robustly expressed in CBC cells. Transcriptome and proteome analyses of Lgr5‐positive intestinal cells define the signature of bona fide intestinal stem cells (ISCs) population. These results offer further insight into the nature of ISCs and will instruct further research on this therapeutically highly relevant topic.