Approximated Gene Expression Trajectories (AGETs) for Gene Regulatory Network Inference on Cell Tracks

The study of pattern formation has benefited from reverse-engineering gene regulatory network (GRN) structure from spatio-temporal quantitative gene expression data. Traditional approaches omit tissue morphogenesis, hence focusing on systems where the timescales of pattern formation and morphogenesis can be separated. In such systems, pattern forms as an emergent property of the underlying GRN. This is not the case in many animal patterning systems, where patterning and morphogenesis are simultaneous. To address pattern formation in these systems we need to adapt our methodologies to explicitly accommodate cell movements and tissue shape changes. In this work we present a novel framework to reverse-engineer GRNs underlying pattern formation in tissues experiencing morphogenetic changes and cell rearrangements. By combination of quantitative data from live and fixed embryos we approximate gene expression trajectories (AGETs) in single cells and use a subset to reverse-engineer candidate GRNs using a Markov Chain Monte Carlo approach. GRN fit is assessed by simulating on cell tracks (live-modelling) and comparing the output to quantitative data-sets. This framework outputs candidate GRNs that recapitulate pattern formation at the level of the tissue and the single cell. To our knowledge, this inference methodology is the first to integrate cell movements and gene expression data, making it possible to reverse-engineer GRNs patterning tissues undergoing morphogenetic changes.

Bmp Signal Gradient Modulates Convergent Cell Movement via Xarhgef3.2 during Gastrulation of Xenopus Embryos

Gastrulation is a critical step in the establishment of a basic body plan during development. Convergence and extension (CE) cell movements organize germ layers during gastrulation. Noncanonical Wnt signaling has been known as major signaling that regulates CE cell movement by activating Rho and Rac. In addition, Bmp molecules are expressed in the ventral side of a developing embryo, and the ventral mesoderm region undergoes minimal CE cell movement while the dorsal mesoderm undergoes dynamic cell movements. This suggests that Bmp signal gradient may affect CE cell movement. To investigate whether Bmp signaling negatively regulates CE cell movements, we performed microarray-based screening and found that the transcription of Xenopus Arhgef3.2 (Rho guanine nucleotide exchange factor) was negatively regulated by Bmp signaling. We also showed that overexpression or knockdown of Xarhgef3.2 caused gastrulation defects. Interestingly, Xarhgef3.2 controlled gastrulation cell movements through interacting with Disheveled (Dsh2) and Dsh2-associated activator of morphogenesis 1 (Daam1). Our results suggest that Bmp gradient affects gastrulation cell movement (CE) via negative regulation of Xarhgef3.2 expression.

The Adaptor Protein Lurap1 Is Required for Cell Cohesion during Epiboly Movement in Zebrafish

Cell adhesion and polarized cellular behaviors play critical roles in a wide variety of morphogenetic events. In the zebrafish embryo, epiboly represents an important process of epithelial morphogenesis that involves differential cell adhesion and dynamic cell shape changes for coordinated movements of different cell populations, but the underlying mechanism remains poorly understood. The adaptor protein Lurap1 functions to link myotonic dystrophy kinase-related Rac/Cdc42-binding kinase with MYO18A for actomyosin retrograde flow in cell migration. We previously reported that it interacts with Dishevelled in convergence and extension movements during gastrulation. Here, we show that it regulates blastoderm cell adhesion and radial cell intercalation during epiboly. In zebrafish mutant embryos with loss of both maternal and zygotic Lurap1 function, deep cell multilayer of the blastoderm exhibit delayed epiboly with respect to the superficial layer. Time-lapse imaging reveals that these deep cells undergo unstable intercalation, which impedes their expansion over the yolk cell. Cell sorting and adhesion assays indicate reduced cellular cohesion of the blastoderm. These defects are correlated with disrupted cytoskeletal organization in the cortex of blastoderm cells. Thus, the present results extend our previous works by demonstrating that Lurap1 is required for cell adhesion and cell behavior changes to coordinate cell movements during epithelial morphogenesis. They provide insights for a further understanding of the regulation of cytoskeletal organization during gastrulation cell movements.

An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia

Introduction Mechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. Methods We show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains. Results As an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). Conclusions This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia.

Temporal integration of inductive cues on the way to gastrulation

Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells’ ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo—which normally only receives a mesoderm-inducing cue—reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.

Twinfilin1 controls lamellipodial protrusive activity and actin turnover during vertebrate gastrulation

The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1 (Twf1). We find that Twf1 is essential for convergent extension, and loss of Twf1 results in a failure of lamellipodial dynamics and polarity. Moreover, Twf1 loss results in a failure to assemble polarized cytoplasmic actin cables essential for convergent extension. These data provide an in vivo complement to our more-extensive understanding of Twf1 action in vitro and provide new links between the core machinery of actin regulation and specialized cell behaviors of embryonic morphogenesis.

Symmetry and fluctuation of cell movements in neural crest-derived facial mesenchyme

In the face, symmetry is established when bilateral streams of neural crest cells leave the neural tube at the same time, follow identical migration routes and then give rise to the facial prominences. However developmental instability exists, particularly surrounding the steps of lip fusion. The causes of instability are unknown but inability to cope with developmental fluctuations are a likely cause of congenital malformations such as non-syndromic orofacial clefts. Here, we tracked cell movements over time in the frontonasal mass which forms the facial midline and participates in lip fusion using live-cell imaging. Our mathematical examination of cell velocity vectors uncovered temporal fluctuations in several parameters including order/disorder, symmetry/asymmetry and divergence/convergence. We found that treatment with a RhoGTPase inhibitor completely disrupted the temporal fluctuations in all measures and blocked morphogenesis. Thus we discovered that genetic control of symmetry extends to mesenchymal cell movements and that these movements are of the type that could be perturbed in in asymmetrical malformations such as non-syndromic cleft lip.