Bomyi Lim

Enhancer Control of Transcriptional Bursting

Transcription is episodic, consisting of a series of discontinuous bursts. Using live-imaging methods and quantitative analysis, we examine transcriptional bursting in living Drosophila embryos. Different developmental enhancers positioned downstream of synthetic reporter genes produce transcriptional bursts with similar amplitudes and duration but generate very different bursting frequencies, with strong enhancers producing more bursts than weak enhancers. Insertion of an insulator reduces the number of bursts and the corresponding level of gene expression, suggesting that enhancer regulation of bursting frequency is a key parameter of gene control in development. We also show that linked reporter genes exhibit coordinated bursting profiles when regulated by a shared enhancer, challenging conventional models of enhancer-promoter looping.

Zelda Potentiates Morphogen Activity by Increasing Chromatin Accessibility

Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis. Here we tested this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds. In agreement with the model, we show that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. We present evidence that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. We propose that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA.

Gene Regulation by MAPK Substrate Competition

Developing tissues are patterned by coordinated activities of signaling systems, which can be integrated by a regulatory region of a gene that binds multiple transcription factors or by a transcription factor that is modified by multiple enzymes. Based on a combination of genetic and imaging experiments in the early Drosophila embryo, we describe a signal integration mechanism that cannot be reduced to a single gene regulatory element or a single transcription factor. This mechanism relies on an enzymatic network formed by mitogen-activated protein kinase (MAPK) and its substrates. Specifically, anteriorly localized MAPK substrates, such as Bicoid, antagonize MAPK-dependent downregulation of Capicua, a repressor that is involved in gene regulation along the dorsoventral axis of the embryo. MAPK substrate competition provides a basis for ternary interaction of the anterior, dorsoventral, and terminal patterning systems. A mathematical model of this interaction can explain gene expression patterns with both anteroposterior and dorsoventral polarities.

Pattern formation by graded and uniform signals in the early Drosophila embryo.

The early Drosophila embryo is patterned by graded distributions of maternal transcription factors. Recent studies revealed that pattern formation by these graded signals depends on uniformly expressed transcriptional activators, such as Zelda. Removal of Zelda influences both the timing and the spatial expression domains for most of the genes controlled by maternal gradients. We demonstrate that some of these patterning defects, which range from temporal delay to loss of expression, can be rationalized with the use of a mathematical model based on cooperative binding of graded and uniform factors. This model makes a number of predictions, which we confirm experimentally by analyzing the expression of short gastrulation (sog), a gene that is controlled by a combination of the Dorsal morphogen gradient and Zelda. The proposed model suggests a general mechanism for the formation of nested gene expression domains, which is a hallmark of tissue patterning by morphogen gradients. According to this mechanism, the differential effects of a morphogen on its target genes can depend on their differential sensitivity to uniform factors.

Microfluidic trap array for massively parallel imaging of Drosophila embryos

Here we describe a protocol for the fabrication and use of a microfluidic device to rapidly orient >700 Drosophila embryos in parallel for end-on imaging. The protocol describes master microfabrication (∼1 d), polydimethylsiloxane molding (few hours), system setup and device operation (few minutes) and imaging (depending on application). Our microfluidics-based approach described here is one of the first to facilitate rapid orientation for end-on imaging, and it is a major breakthrough for quantitative studies on Drosophila embryogenesis. The operating principle of the embryo trap is based on passive hydrodynamics, and it does not require direct manipulation of embryos by the user; biologists following the protocol should be able to repeat these procedures. The compact design and fabrication materials used allow the device to be used with traditional microscopy setups and do not require specialized fixtures. Furthermore, with slight modification, this array can be applied to the handling of other model organisms and oblong objects.

Kinetics of gene derepression by ERK signaling

ERK controls gene expression in development, but mechanisms that link ERK activation to changes in transcription are not well understood. We used high-resolution analysis of signaling dynamics to study transcriptional interpretation of ERK signaling during Drosophila embryogenesis, at a stage when ERK induces transcription of intermediate neuroblasts defective (ind), a gene essential for patterning of the nerve cord. ERK induces ind by antagonizing its repression by Capicua (Cic), a transcription factor that acts as a sensor of receptor tyrosine kinases in animal development and human diseases. A recent study established that active ERK reduces the nuclear levels of Cic, but it remained unclear whether this is required for the induction of Cic target genes. We provide evidence that Cic binding sites within the regulatory DNA of ind control the spatial extent and the timing of ind expression. At the same time, we demonstrate that ERK induces ind before Cic levels in the nucleus are reduced. Based on this, we propose that ERK-dependent relief of gene repression by Cic is a two-step process, in which fast reduction of repressor activity is followed by slower changes in nuclear localization and overall protein levels. This may be a common feature of systems in which ERK induces genes by relief of transcriptional repression.

Dynamics of Inductive ERK Signaling in the Drosophila Embryo

Transient activation of the highly conserved extracellular-signal-regulated kinase (ERK) establishes precise patterns of cell fates in developing tissues. Quantitative parameters of these transients are essentially unknown, but a growing number of studies suggest that changes in these parameters can lead to a broad spectrum of developmental abnormalities. We provide a detailed quantitative picture of an ERK-dependent inductive signaling event in the early Drosophila embryo, an experimental system that offers unique opportunities for high-throughput studies of developmental signaling. Our analysis reveals a spatiotemporal pulse of ERK activation that is consistent with a model in which transient production of a short-ranged ligand feeds into a simple signal interpretation system. The pulse of ERK signaling acts as a switch in controlling the expression of the ERK target gene. The quantitative approach that led to this model, based on the integration of data from fixed embryos and live imaging, can be extended to other developmental systems patterned by transient inductive signals.

Temporal ordering and registration of images in studies of developmental dynamics

Progress of development is commonly reconstructed from imaging snapshots of chemical or mechanical processes in fixed tissues. As a first step in these reconstructions, snapshots must be spatially registered and ordered in time. Currently, image registration and ordering are often done manually, requiring a significant amount of expertise with a specific system. However, as the sizes of imaging data sets grow, these tasks become increasingly difficult, especially when the images are noisy and the developmental changes being examined are subtle. To address these challenges, we present an automated approach to simultaneously register and temporally order imaging data sets. The approach is based on vector diffusion maps, a manifold learning technique that does not require a priori knowledge of image features or a parametric model of the developmental dynamics. We illustrate this approach by registering and ordering data from imaging studies of pattern formation and morphogenesis in three model systems. We also provide software to aid in the application of our methodology to other experimental data sets. Summary: Learning algorithms allow developmental dynamics to be reconstructed through the automatic registration and ordering of fixed images of developing tissues.

RTK signaling modulates the Dorsal gradient

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. Here, we demonstrate that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, we show that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, our results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo.

Effect of composition on water permeability of model stratum corneum lipid membranes

The stratum corneum (SC), composed of corneocytes and intercellular lipid membranes, is the outermost layer of the epidermis, and its main function is the regulation of water loss from the skin. The major components of the SC lipid membranes are ceramides (CER), cholesterol (CHOL), and free fatty acids (FFA), which are organized in multilamellar structures between corneocytes. The intercellular SC lipid membrane is believed to provide the main pathway for the transport of water and other substances through the skin. While changes in the composition of the SC lipid membranes have been shown to affect the organization of the lipid molecules, little is known about the effect of compositional changes on their water permeability. In this work, we study the effect of membrane composition on the water permeability of model SC lipid membranes using a quartz crystal microbalance with dissipation monitoring (QCM-D). The QCM-D method enables the direct determination of the diffusivity (D), solubility (S), and permeability (P) of water through the model SC lipid membranes. We find that D and S weakly depend on the chain length of saturated fatty acids, while P shows no significant dependence. In contrast, the saturation level of free fatty acids and the structure of ceramide have significant influence on D and S, respectively, resulting in significant changes in P. By taking advantage of the dissipation monitoring capability of the QCM-D at multiple overtones, we find that the shear modulus (G) of the SC lipid membranes depends on its composition and decreases upon water absorption by the membranes.