Biology subjects

Shvartsman, S. Y.

Publications and source records attributed to Shvartsman, S. Y..

14 recordsLinked to original sources

bioRxiv2025

A model for boundary-driven tissue morphogenesis

Tissue deformations during morphogenesis can be active, driven by internal processes, or passive, resulting from stresses applied at their boundaries. Here, we introduce the Drosophila hindgut primordium as a model for studying boundary-driven tissue morphogenesis. We characterize its deformations and show that its complex shape changes can be a passive consequence of the deformations of the active regions of the embryo that surround it. First, we find an intermediate characteristic triangular shape in the 3D deformations of the hindgut. We construct a minimal model of the hindgut primordium as an elastic ring deformed by active midgut invagination and germ band extension on an ellipsoidal surface, which robustly captures the symmetry-breaking into this triangular shape. We then quantify the 3D kinematics of the tissue by a set of contours and discover that the hindgut deforms in two stages: an initial translation on the curved embryo surface followed by a rapid breaking of shape symmetry. We extend our model to show that the contour kinematics in both stages are consistent with our passive picture. Our results suggest that the role of in-plane deformations during hindgut morphogenesis is to translate the tissue to a region with anisotropic embryonic curvature and show that uniform boundary conditions are sufficient to generate the observed nonuniform shape change. Our work thus provides a possible explanation for the various characteristic shapes of blastopore-equivalents in different organisms and a framework for the mechanical emergence of global morphologies in complex developmental systems.

biophysics↗

bioRxiv2025

In vivo measurements of receptor tyrosine kinase activity reveal feedback regulation of a developmental gradient

A lack of tools for detecting receptor activity in vivo has limited our ability to fully explore receptor-level control of developmental patterning. Here, we extend a new class of biosensors for receptor tyrosine kinase (RTK) activity, the pYtag system, to visualize endogenous RTK activity in Drosophila. We build biosensors for three Drosophila RTKs that function across developmental stages and tissues. By characterizing Torso::pYtag during terminal patterning in the early embryo, we find that Torso activity differs from downstream ERK activity in two surprising ways: Torso activity is narrowly restricted to the poles but produces a broader gradient of ERK, and Torso activity decreases over developmental time while ERK activity is sustained. This decrease in Torso activity is driven by ERK pathway-dependent negative feedback. Our results suggest an updated model of terminal patterning where a narrow domain of Torso activity, tuned in amplitude by negative feedback, locally activates signaling effectors which diffuse through the syncytial embryo to form the ERK gradient. Altogether, this work highlights the usefulness of pYtags for investigating receptor-level regulation of developmental patterning.

developmental biology↗

bioRxiv2024

Disrupted developmental signaling induces novel transcriptional states

Signaling pathways induce stereotyped transcriptional changes as stem cells progress into mature cell types during embryogenesis. Signaling perturbations are necessary to discover which genes are responsive or insensitive to pathway activity. However, gene regulation is additionally dependent on cell state-specific factors like chromatin modifications or transcription factor binding. Thus, transcriptional profiles need to be assayed in single cells to identify potentially multiple, distinct perturbation responses among heterogeneous cell states in an embryo. In perturbation studies, comparing heterogeneous transcriptional states among experimental conditions often requires samples to be collected over multiple independent experiments. Datasets produced in such complex experimental designs can be confounded by batch effects. We present Design-Aware Integration of Single Cell ExpEriments (DAISEE), a new algorithm that models perturbation responses in single-cell datasets with a complex experimental design. We demonstrate that DAISEE improves upon a previously available integrative non-negative matrix factorization framework, more efficiently separating perturbation responses from confounding variation. We use DAISEE to integrate newly collected single-cell RNA-sequencing datasets from 5-hour old zebrafish embryos expressing optimized photoswitchable MEK (psMEK), which globally activates the extracellular signal-regulated kinase (ERK), a signaling molecule involved in many cell specification events. psMEK drives some cells that are normally not exposed to ERK signals towards other wild type states and induces novel states expressing a mixture of transcriptional programs, including precociously activated endothelial genes. ERK signaling is therefore capable of introducing profoundly new gene expression states in developing embryos. Significance StatementSignaling perturbations produce heterogeneous transcriptional responses that must be measured at the single-cell level. Data integration techniques allow us to model these responses which, however, can be confounded by batch effects. We present a computational tool (DAISEE) for extracting the common and perturbation-specific features of single-cell datasets representing multiple experimental conditions while achieving efficient batch effect correction. DAISEE outperforms its predecessor and will enable accurate analysis of a broad range of single-cell datasets. DAISEE applied to new single-cell RNA sequencing data from zebrafish embryos shows that gain-of-function signaling perturbations can induce novel states. Our analysis suggests that a wild type endothelial cell-specification program can be activated in abnormal developmental contexts when the extracellular signal-regulated kinase (ERK) pathway is deregulated.

developmental biology↗

bioRxiv2024

ERK inhibits Cic repressor function via multisite phosphorylation

The receptor tyrosine kinase (RTK)/Extracellular Signal-Regulated Kinase (ERK) signaling pathway controls cell proliferation, differentiation, and survival. How ERK activation is relayed to its phosphorylation targets is not well understood. The transcriptional repressor Capicua (Cic) has emerged as a key target for ERK-mediated downregulation in Drosophila and mammals, and mutations in human CIC result in cancer and neurological diseases. Phosphorylation by ERK is critical for Cic downregulation, but the identities of phosphosites in Drosophila Cic are unknown. Here, we identify sites of phosphorylation in Cic that are directly targeted by ERK and validate their developmental functions in vivo using mutant Cic variants. Cic phosphosites are distributed throughout the length of the protein, and a group of centrally located sites appears to have a primary role in Cic downregulation. Cic mutated in 20 high-confidence sites behaves as a "super-repressor" in vivo that is largely insensitive to ERK-mediated downregulation, despite fully retaining the ability to bind to ERK. No single site is sufficient to turn off Cic activity; instead, we find that ERK must phosphorylate multiple sites in Cic simultaneously to achieve full downregulation. This multisite phosphorylation likely targets phosphodegrons that are recognized by ubiquitin ligases such as Ago/FBXW7 and contributes to Cic degradation. This study advances our understanding of the molecular mechanisms of signal interpretation downstream of the RTK/ERK signaling network.

developmental biology↗

bioRxiv2023

Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo

Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be blurred through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.

developmental biology↗

bioRxiv2023

Evolutionary history of MEK1 illuminates the nature of cancer and RASopathy mutations

Mutations in signal transduction pathways lead to various diseases including cancers. MEK1 kinase, encoded by the human MAP2K1 gene, is one of the central components of the MAPK pathway and more than a hundred somatic mutations in MAP2K1 gene were identified in various tumors. Germline mutations deregulating MEK1 also lead to congenital abnormalities, such as the Cardiofaciocutaneous Syndrome and Arteriovenous Malformation. Evaluating variants associated with a disease is a challenge and computational genomic approaches aid in this process. Establishing evolutionary history of a gene improves computational prediction of disease-causing mutations; however, the evolutionary history of MEK1 is not well understood. Here, by revealing a precise evolutionary history of MEK1 we construct a well-defined dataset of MEK1 metazoan orthologs, which provides sufficient depth to distinguish between conserved and variable amino acid positions. We used this dataset to match known and predicted disease-causing and benign mutations to evolutionary changes observed in corresponding amino acid positions. We found that all known and the vast majority of suspected disease-causing mutations are evolutionarily intolerable. We selected several MEK1 mutations that cannot be unambiguously assessed by automated variant prediction tools, but that are confidently identified as evolutionary intolerant and thus "damaging" by our approach, for experimental validation in Drosophila. In all cases, evolutionary intolerant variants caused increased mortality and severe defects in fruit fly embryos confirming their damaging nature predicted by out computational strategy. We anticipate that our analysis will serve as a blueprint to help evaluate known and novel missense variants in MEK1 and that our approach will contribute to improving automated tools for disease-associated variant interpretation. Significance StatementHigh-throughput genome sequencing has significantly improved diagnosis, management, and treatment of genetic diseases and cancers. However, in addition to its indisputable utility, genome sequencing produces many variants that cannot be easily interpreted - so called variants of uncertain significance (VUS). Various automated bioinformatics tools can help predicting functional consequences of VUS, but their accuracy is relatively low. Here, by tracing precise evolutionary history of each amino acid position in MEK1 kinase, mutations in which cause neurodegenerative diseases and cancer in humans, we can establish whether VUS seen in humans are evolutionarily tolerant. Using published data and newly performed experiments in an animal model, we show that evolutionarily tolerable variants in MEK1 are benign, whereas intolerable substitutions are damaging. Our approach will help in diagnostics of MEK1-associated diseases, it is generalizable to many other disease-associated genes, and it can help improving automated predictors.

evolutionary biology↗

bioRxiv2022

Sfrp2 is a multifunctional regulator of rodent color patterns

Animal pigment patterns are excellent models to elucidate mechanisms of biological organization. Although theoretical simulations, such as Turing reaction-diffusion systems, recapitulate many animal patterns, they are insufficient to account for those showing a high degree of spatial organization and reproducibility. Here, we compare the coats of the African striped mouse (Rhabdomys pumilio) and the laboratory mouse (Mus musculus) to study the molecular mechanisms controlling stripe pattern formation. By combining transcriptomics, mathematical modeling, and mouse transgenics, we show that Sfrp2 regulates the distribution of hair follicles and establishes an embryonic prepattern that foreshadows pigment stripes. Moreover, by developing and employing in vivo gene editing experiments in striped mice, we find that Sfrp2 knockout is sufficient to alter the stripe pattern. Strikingly, mutants also exhibit changes in coat color, revealing an additional function of Sfrp2 in regulating hair color. Thus, a single factor controls coat pattern formation by acting both as an orienting signaling mechanism and a modulator of pigmentation. By uncovering a multifunctional regulator of stripe formation, our work provides insights into the mechanisms by which spatial patterns are established in developing embryos and the molecular basis of phenotypic novelty.

developmental biology↗

bioRxiv2021

ERK signaling dissolves ERF Repression Condensates in Living Embryos

Phase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor ERF, which has been linked to various human developmental disorders. Using high resolution imaging we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during the cell cycle, providing the first visualization of a nuclear phase separation process that is regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell-cell signaling events.

developmental biology↗

bioRxiv2021

Deconstructing Gastrulation at the Single Cell Level

Gastrulation movements in all animal embryos start with regulated deformations of patterned epithelial sheets. Current studies of gastrulation use a wide range of model organisms and emphasize either large-scale tissue processes or dynamics of individual cells and cell groups 1,2,11-13,3-10. Here we take a step towards bridging these complementary strategies and deconstruct early stages of gastrulation in the entire Drosophila embryo, where transcriptional patterns in the blastoderm give rise to region-specific cell behaviors. Our approach relies on an integrated computational framework for cell segmentation and tracking and on efficient algorithms for event detection. Our results reveal how thousands of cell shape changes, divisions, and intercalations drive large-scale deformations of the patterned blastoderm, setting the stage for systems-level dissection of a pivotal step in animal development.

developmental biology↗

bioRxiv2021

From heterogeneous datasets to predictive models of embryonic development

Modern studies of embryogenesis are increasingly quantitative, powered by rapid advances in imaging, sequencing, and genome manipulation technologies. Deriving mechanistic insights from the complex datasets generated by these new tools requires systematic approaches for data-driven analysis of the underlying developmental processes. Here we use data from our work on signal-dependent gene repression in the Drosophila embryo to illustrate how computational models can compactly summarize quantitative results of live imaging, chromatin immunoprecipitation, and optogenetic perturbation experiments. The presented computational approach is ideally suited for integrating rapidly accumulating quantitative data and for guiding future studies of embryogenesis.

developmental biology↗

bioRxiv2020

Evaluating the Simple Arrhenius Equation for the Temperature Dependence of Complex Developmental Processes

The famous Arrhenius equation is well motivated to describe 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 multistep biological processes, using frog and fruit fly embryogenesis as two canonical models. We find the Arrhenius equation provides a good approximation for the temperature dependence of embryogenesis, even though individual developmental stages scale differently with temperature. At low and high temperatures, however, we observed significant departures from idealized Arrhenius Law behavior. When we model multistep reactions of idealized chemical networks we are unable to generate comparable deviations from linearity. In contrast, we find the single enzyme GAPDH shows 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 Law and propose that the observed departure from this law results primarily from non-idealized individual steps rather than the complexity of the system.

systems biology↗

bioRxiv2019

Optogenetic rescue of a developmental patterning mutant

Animal embryos are partitioned into spatial domains by complex patterns of signaling and gene expression, yet it is still largely unknown what features of a developmental signal are essential. Part of the challenge arises because it has been impossible to \"paint\" arbitrary signaling patterns on an embryo to test their sufficiency. Here we demonstrate exactly this capability by combining optogenetic control of Ras/Erk signaling with the genetic loss of terminal signaling in early Drosophila embryos. Simple all-or-none light inputs at the embryonic termini were able to completely rescue normal development, generating viable larvae and fertile adults from this otherwise-lethal genetic mutant. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered by different cumulative doses of Erk. These results open the door to the targeted design of complex morphogenetic outcomes as well as the ability to correct patterning errors that underlie developmental defects.\n\nSummarySpatial gradients of protein activity pattern all developing embryos. But which features of these patterns are essential and which are dispensable for development? Answering this question has been deceptively difficult, due to the difficulty of shaping spatial gradients using chemical or genetic perturbations. Johnson and colleagues use optogenetics to fully restore normal embryo development with a pattern of light, opening the door to complete control over spatial organization in developing and engineered tissues.

developmental biology↗

bioRxiv2018

Signaling dynamics control cell fate in the early Drosophila embryo

The Erk mitogen-activated protein kinase plays diverse roles in animal development, where its activity is associated with phenomena including cell migration, proliferation and differentiation. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? Here, we combine precise optogenetic control with genetic perturbations to dissect Erk-dependent cellular responses in the early Drosophila embryo. We find that light-stimulated Erk activity is sufficient to posterior-ize the majority of the embryo, leading to massive apical constriction through expression of the autocrine receptor-ligand pair mist and fog. Ectopic contraction at non-terminal positions requires at least 1 h of high-amplitude Erk signaling, whereas a 30 min pulse of Erk activity patterns non-contractile neurogenic ectoderm during the same time window. In contrast to the canonical transient versus sustained model, the cell fate switch is triggered by the cumulative load of Erk signaling, not the duration of a single persistent pulse. Our results reveal that the early fly embryo harbors a classic example of dynamic cell fate control, where the total dose of Erk activity selects between two distinct physiological outcomes.

developmental biology↗

bioRxiv2017

Synthesizing developmental trajectories

Dynamical processes in biology are studied using an ever-increasing number of techniques, each of which brings out unique features of the system. One of the current challenges is to develop systematic approaches for fusing heterogeneous datasets into an integrated view of multivariable dynamics. We demonstrate that heterogeneous data fusion can be successfully implemented within a semi-supervised learning framework that exploits the intrinsic geometry of high-dimensional datasets. We illustrate our approach using a dataset from studies of pattern formation in Drosophila. The result is a continuous trajectory that reveals the joint dynamics of gene expression, subcellular protein localization, protein phosphorylation, and tissue morphogenesis. Our approach can be readily adapted to other imaging modalities and forms a starting point for further steps of data analytics and modeling of biological dynamics.

developmental biology↗