Optimal Decoding of Cellular Identities in a Genetic Network.

In developing organisms, spatially prescribed cell identities are thought to be determined by the expression levels of multiple genes. Quantitative tests of this idea, however, require a theoretical framework capable of exposing the rules and precision of cell specification over developmental time. We use the gap gene network in the early fly embryo as an example to show how expression levels of the four gap genes can be jointly decoded into an optimal specification of position with 1% accuracy. The decoder correctly predicts, with no free parameters, the dynamics of pair-rule expression patterns at different developmental time points and in various mutant backgrounds. Precise cellular identities are thus available at the earliest stages of development, contrasting the prevailing view of positional information being slowly refined across successive layers of the patterning network. Our results suggest that developmental enhancers closely approximate a mathematically optimal decoding strategy.

Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition.

A conserved feature of the midblastula transition (MBT) is a requirement for a functional DNA replication checkpoint to coordinate cell-cycle remodeling and zygotic genome activation (ZGA). We have investigated what triggers this checkpoint during Drosophila embryogenesis. We find that the magnitude of the checkpoint scales with the quantity of transcriptionally engaged DNA. Measuring RNA polymerase II (Pol II) binding at 20 min intervals over the course of ZGA reveals that the checkpoint coincides with widespread de novo recruitment of Pol II that precedes and does not require a functional checkpoint. This recruitment drives slowing or stalling of DNA replication at transcriptionally engaged loci. Reducing Pol II recruitment in zelda mutants both reduces replication stalling and bypasses the requirement for a functional checkpoint. This suggests a model where the checkpoint functions as a feedback mechanism to remodel the cell cycle in response to nascent ZGA.

The Heidelberg Screen for Pattern Mutants of Drosophila: A Personal Account.

In large-scale mutagenesis screens performed in 1979-1980 at the EMBL in Heidelberg, we isolated mutations affecting the pattern or structure of the larval cuticle in Drosophila. The 600 mutants we characterized could be assigned to 120 genes and represent the majority of such genes in the genome. These mutants subsequently provided a rich resource for understanding many fundamental developmental processes, such as the transcriptional hierarchies controlling segmentation, the establishment of cell states by signaling pathways, and the differentiation of epithelial cells. Most of the Heidelberg genes are now molecularly known, and many of them are conserved in other animals, including humans. Although the screens were initially driven entirely by curiosity, the mutants now serve as models for many human diseases. In this review, we describe the rationale of the screening procedures and provide a classification of the genes on the basis of their initial phenotypes and the subsequent molecular analyses.

Dynamics of Drosophila endoderm specification.

During early Drosophila embryogenesis, a network of gene regulatory interactions orchestrates terminal patterning, playing a critical role in the subsequent formation of the gut. We utilized CRISPR gene editing at endogenous loci to create live reporters of transcription and light-sheet microscopy to monitor the individual components of the posterior gut patterning network across 90 min prior to gastrulation. We developed a computational approach for fusing imaging datasets of the individual components into a common multivariable trajectory. Data fusion revealed low intrinsic dimensionality of posterior patterning and cell fate specification in wild-type embryos. The simple structure that we uncovered allowed us to construct a model of interactions within the posterior patterning regulatory network and make testable predictions about its dynamics at the protein level. The presented data fusion strategy is a step toward establishing a unified framework that would explore how stochastic spatiotemporal signals give rise to highly reproducible morphogenetic outcomes.

Trading bits in the readout from a genetic network.

In the regulation of gene expression, information of relevance to the organism is represented by the concentrations of transcription factor molecules. To extract this information the cell must effectively "measure" these concentrations, but there are physical limits to the precision of these measurements. We use the gap gene network in the early fly embryo as an example of the tradeoff between the precision of concentration measurements and the transmission of relevant information. For thresholded measurements we find that lower thresholds are more important, and fine tuning is not required for near-optimal information transmission. We then consider general sensors, constrained only by a limit on their information capacity, and find that thresholded sensors can approach true information theoretic optima. The information theoretic approach allows us to identify the optimal sensor for the entire gap gene network and to argue that the physical limitations of sensing necessitate the observed multiplicity of enhancer elements, with sensitivities to combinations rather than single transcription factors.

Temperature-Induced uncoupling of cell cycle regulators.

The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness we examined cell cycle progression in early Drosophila embryos at different temperatures. Our experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 â€‹°C), the relative duration of individual steps varies with temperature. We find that the transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, we quantified the ratio of activities of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.

Evaluating the Arrhenius equation for developmental processes.

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 ß-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.

Template-based mapping of dynamic motifs in tissue morphogenesis.

Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. Here, we present a "template-based search" approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. We formulated the task of motif mapping as a subsequence matching problem and solved it using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows our algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate our approach, we mapped cell intercalations during germband extension in the early Drosophila embryo. Our framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. Our formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.

Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation.

Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is used throughout development in most animals. Little is known, however, about how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. Here we apply particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow formation. We find that cytoplasmic redistribution during the lengthening phase of ventral furrow formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to, or driving force on, the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells before gastrulation ('acellular' embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild-type embryos. Our results indicate that during the lengthening phase of ventral furrow formation, hydrodynamic behaviour of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable.

Independent active and thermodynamic processes govern the nucleolus assembly in vivo.

Membraneless organelles play a central role in the organization of protoplasm by concentrating macromolecules, which allows efficient cellular processes. Recent studies have shown that, in vitro, certain components in such organelles can assemble through phase separation. Inside the cell, however, such organelles are multicomponent, with numerous intermolecular interactions that can potentially affect the demixing properties of individual components. In addition, the organelles themselves are inherently active, and it is not clear how the active, energy-consuming processes that occur constantly within such organelles affect the phase separation behavior of the constituent macromolecules. Here, we examine the phase separation model for the formation of membraneless organelles in vivo by assessing the two features that collectively distinguish it from active assembly, namely temperature dependence and reversibility. We use a microfluidic device that allows accurate and rapid manipulation of temperature and examine the quantitative dynamics by which six different nucleolar proteins assemble into the nucleoli of Drosophila melanogaster embryos. Our results indicate that, although phase separation is the main mode of recruitment for four of the studied proteins, the assembly of the other two is irreversible and enhanced at higher temperatures, behaviors indicative of active recruitment to the nucleolus. These two subsets of components differ in their requirements for ribosomal DNA; the two actively assembling components fail to assemble in the absence of ribosomal DNA, whereas the thermodynamically driven components assemble but lose temporal and spatial precision.

Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids.

Many models of morphogenesis are forced to assume specific mechanical properties of cells, because the actual mechanical properties of living tissues are largely unknown. Here, we measure the rheology of epithelial cells in the cellularizing Drosophila embryo by injecting magnetic particles and studying their response to external actuation. We establish that, on timescales relevant to epithelial morphogenesis, the cytoplasm is predominantly viscous, whereas the cellular cortex is elastic. The timescale of elastic stress relaxation has a lower bound of 4 min, which is comparable to the time required for internalization of the ventral furrow during gastrulation. The cytoplasm was measured to be ∼103-fold as viscous as water. We show that elasticity depends on the actin cytoskeleton and conclude by discussing how these results relate to existing mechanical models of morphogenesis.

Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity.

Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows.

Polarity protein Par3/Bazooka follows myosin-dependent junction repositioning.

The polarity protein Par3/Bazooka (Baz) has been established as a central component of the apical basal polarity system that determines the position of cell-cell junctions in epithelial cells. Consistent with that view, we show that shortly before gastrulation in Drosophila, Baz protein in the mesoderm is down-regulated from junctional sites in response to Snail (Sna) expression. This down-regulation leads to a specific decrease in adherens junctions without affecting other E-Cadherin pools. However, we further show that, interactions between Baz and junctions are not unidirectional. During apical constriction and the internalization of the mesoderm, down-regulation of Baz is transiently blocked as adherens junctions shift apically and are strengthened in response to tension generated by contractile actomyosin. When such junction remodeling is prevented by down-regulating myosin, Baz is lost prematurely in mesodermal epithelium. During such apical shifts, Baz is initially left behind as the junction shifts position, but then re-accumulates at the new location of the junctions. On the dorsal side of the embryo, a similar pattern of myosin activity appears to limit the basal shift in junctions normally driven by Baz that controls epithelium folding. Our results suggest a model where the sensitivity of Baz to Sna expression leads to the Sna-dependent junction disassembly required for a complete epithelium-mesenchymal transition. Meanwhile this loss of Baz-dependent junction maintenance is countered by the myosin-based mechanism which promotes an apical shift and strengthening of junctions accompanied by a transient re-positioning and maintenance of Baz proteins.

Diversity of epithelial morphogenesis during eggshell formation in drosophilids.

The eggshells of drosophilid species provide a powerful model for studying the origins of morphological diversity. The dorsal appendages, or respiratory filaments, of these eggshells display a remarkable interspecies variation in number and shape, and the epithelial patterning underlying the formation of these structures is an area of active research. To extend the analysis of dorsal appendage formation to include morphogenesis, we developed an improved 3D image reconstruction approach. This approach revealed considerable interspecies variation in the cell shape changes and neighbor exchanges underlying appendage formation. Specifically, although the appendage floor in Drosophila melanogaster is formed through spatially ordered neighbor exchanges, the same structure in Scaptodrosophila pattersoni is formed through extreme changes in cell shape, whereas Drosophila funebris appears to display a combination of both cellular mechanisms. Furthermore, localization patterns of Par3/Bazooka suggest a self-organized, cell polarity-based origin for the variability of appendage number in S. pattersoni. Our results suggest that species deploy different combinations of apically and basally driven mechanisms to convert a two-dimensional primordium into a three-dimensional structure, and provide new directions for exploring the molecular origins of interspecies morphological variation.

Differential positioning of adherens junctions is associated with initiation of epithelial folding.

During tissue morphogenesis, simple epithelial sheets undergo folding to form complex structures. The prevailing model underlying epithelial folding involves cell shape changes driven by myosin-dependent apical constriction. Here we describe an alternative mechanism that requires differential positioning of adherens junctions controlled by modulation of epithelial apical-basal polarity. Using live embryo imaging, we show that before the initiation of dorsal transverse folds during Drosophila gastrulation, adherens junctions shift basally in the initiating cells, but maintain their original subapical positioning in the neighbouring cells. Junctional positioning in the dorsal epithelium depends on the polarity proteins Bazooka and Par-1. In particular, the basal shift that occurs in the initiating cells is associated with a progressive decrease in Par-1 levels. We show that uniform reduction of the activity of Bazooka or Par-1 results in uniform apical or lateral positioning of junctions and in each case dorsal fold initiation is abolished. In addition, an increase in the Bazooka/Par-1 ratio causes formation of ectopic dorsal folds. The basal shift of junctions not only alters the apical shape of the initiating cells, but also forces the lateral membrane of the adjacent cells to bend towards the initiating cells, thereby facilitating tissue deformation. Our data thus establish a direct link between modification of epithelial polarity and initiation of epithelial folding.

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts.

Positional information, in bits.

Cells in a developing embryo have no direct way of "measuring" their physical position. Through a variety of processes, however, the expression levels of multiple genes come to be correlated with position, and these expression levels thus form a code for "positional information." We show how to measure this information, in bits, using the gap genes in the Drosophila embryo as an example. Individual genes carry nearly two bits of information, twice as much as would be expected if the expression patterns consisted only of on/off domains separated by sharp boundaries. Taken together, four gap genes carry enough information to define a cell's location with an error bar of ~1 along the anterior/posterior axis of the embryo. This precision is nearly enough for each cell to have a unique identity, which is the maximum information the system can use, and is nearly constant along the length of the embryo. We argue that this constancy is a signature of optimality in the transmission of information from primary morphogen inputs to the output of the gap gene network.

Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

Heterochromatin represents a significant portion of eukaryotic genomes and has essential structural and regulatory functions. Its molecular organization is largely unknown due to difficulties in sequencing through and assembling repetitive sequences enriched in the heterochromatin. Here we developed a novel strategy using chromosomal rearrangements and embryonic phenotypes to position unmapped Drosophila melanogaster heterochromatic sequence to specific chromosomal regions. By excluding sequences that can be mapped to the assembled euchromatic arms, we identified sequences that are specific to heterochromatin and used them to design heterochromatin specific probes ("H-probes") for microarray. By comparative genomic hybridization (CGH) analyses of embryos deficient for each chromosome or chromosome arm, we were able to map most of our H-probes to specific chromosome arms. We also positioned sequences mapped to the second and X chromosomes to finer intervals by analyzing smaller deletions with breakpoints in heterochromatin. Using this approach, we were able to map >40% (13.9 Mb) of the previously unmapped heterochromatin sequences assembled by the whole-genome sequencing effort on arm U and arm Uextra to specific locations. We also identified and mapped 110 kb of novel heterochromatic sequences. Subsequent analyses revealed that sequences located within different heterochromatic regions have distinct properties, such as sequence composition, degree of repetitiveness, and level of underreplication in polytenized tissues. Surprisingly, although heterochromatin is generally considered to be transcriptionally silent, we detected region-specific temporal patterns of transcription in heterochromatin during oogenesis and early embryonic development. Our study provides a useful approach to elucidate the molecular organization and function of heterochromatin and reveals region-specific variation of heterochromatin.

Torso RTK controls Capicua degradation by changing its subcellular localization.

The transcriptional repressor Capicua (Cic) controls multiple aspects of Drosophila embryogenesis and has been implicated in vertebrate development and human diseases. Receptor tyrosine kinases (RTKs) can antagonize Cic-dependent gene repression, but the mechanisms responsible for this effect are not fully understood. Based on genetic and imaging studies in the early Drosophila embryo, we found that Torso RTK signaling can increase the rate of Cic degradation by changing its subcellular localization. We propose that Cic is degraded predominantly in the cytoplasm and show that Torso reduces the stability of Cic by controlling the rates of its nucleocytoplasmic transport. This model accounts for the experimentally observed spatiotemporal dynamics of Cic in the early embryo and might explain RTK-dependent control of Cic in other developmental contexts.

Pulsed contractions of an actin-myosin network drive apical constriction.

Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. Here we show, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. Our results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally.