Angelike Stathopoulos

Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration

The concerted movement of cells from different germ layers contributes to morphogenesis during early embryonic development. Using an optimized imaging approach and quantitative methods, we analyzed the trajectories of hundreds of ectodermal cells and internalized mesodermal cells within Drosophila embryos over 2 hours during gastrulation. We found a high level of cellular organization, with mesoderm cell movements correlating with some but not all ectoderm movements. During migration, the mesoderm population underwent two ordered waves of cell division and synchronous cell intercalation, and cells at the leading edge stably maintained position. Fibroblast growth factor (FGF) signaling guides mesodermal cell migration; however, we found some directed dorsal migration in an FGF receptor mutant, which suggests that additional signals are involved. Thus, decomposing complex cellular movements can provide detailed insights into collective cell migration.

Promoter-proximal tethering elements regulate enhancer-promoter specificity in the Drosophila Antennapedia complex

Insulator DNAs and promoter competition regulate enhancer–promoter interactions within complex genetic loci. Here we provide evidence for a third mechanism: promoter-proximal tethering elements. The Scr-ftz region of the Antennapedia gene complex includes two known enhancers, AE1 and T1. AE1 selectively interacts with the ftz promoter to maintain pair-rule stripes of ftz expression during gastrulation and germ-band elongation. The T1 enhancer, located 3′ of the ftz gene and ≈25 kb 5′ of the Scr promoter, selectively activates Scr expression in the prothorax and posterior head segments. A variety of P element minigenes were examined in transgenic embryos to determine the basis for specific AE1-ftz and T1-Scr interactions. A 450-bp DNA fragment located ≈100 bp 5′ of the Scr transcription start site is essential for T1-Scr interactions and can mediate long-range activation of a ftz/lacZ reporter gene when placed 5′ of the ftz promoter. We suggest that the Scr450 fragment contains tethering elements that selectively recruit T1 to the Scr promoter. Tethering elements might regulate enhancer–promoter interactions at other complex genetic loci.

Design flexibility in cis-regulatory control of gene expression: Synthetic and comparative evidence

In early Drosophila embryos, the transcription factor Dorsal regulates patterns of gene expression and cell fate specification along the dorsal-ventral axis. How gene expression is produced within the broad lateral domain of the presumptive neurogenic ectoderm is not understood. To investigate transcriptional control during neurogenic ectoderm specification, we examined divergence and function of an embryonic cis-regulatory element controlling the gene short gastrulation (sog). While transcription factor binding sites are not completely conserved, we demonstrate that these sequences are bona fide regulatory elements, despite variable regulatory architecture. Mutation of conserved sequences revealed that putative transcription factor binding sites for Dorsal and Zelda, a ubiquitous maternal transcription factor, are required for proper sog expression. When Zelda and Dorsal sites are paired in a synthetic regulatory element, broad lateral expression results. However, synthetic regulatory elements that contain Dorsal and an additional activator also drive expression throughout the neurogenic ectoderm. Our results suggest that interaction between Dorsal and Zelda drives expression within the presumptive neurogenic ectoderm, but they also demonstrate that regulatory architecture directing expression in this domain is flexible. We propose a model for neurogenic ectoderm specification in which gene regulation occurs at the intersection of temporal and spatial transcription factor inputs.

FGF ligands in Drosophila have distinct activities required to support cell migration and differentiation

Fibroblast growth factor (FGF) signaling controls a vast array of biological processes including cell differentiation and migration, wound healing and malignancy. In vertebrates, FGF signaling is complex, with over 100 predicted FGF ligand-receptor combinations. Drosophila melanogaster presents a simpler model system in which to study FGF signaling, with only three ligands and two FGF receptors (FGFRs) identified. Here we analyze the specificity of FGFR [Heartless (Htl) and Breathless (Btl)] activation by each of the FGF ligands [Pyramus (Pyr), Thisbe (Ths) and Branchless (Bnl)] in Drosophila. We confirm that both Pyr and Ths can activate Htl, and that only Bnl can activate Btl. To examine the role of each ligand in supporting activation of the Htl FGFR, we utilize genetic approaches that focus on the earliest stages of embryonic development. When pyr and ths are equivalently expressed using the Gal4 system, these ligands support qualitatively different FGFR signaling responses. Both Pyr and Ths function in a non-autonomous fashion to support mesoderm spreading during gastrulation, but Pyr exhibits a longer functional range. pyr and ths single mutants exhibit defects in mesoderm spreading during gastrulation, yet only pyr mutants exhibit severe defects in dorsal mesoderm specification. We demonstrate that the Drosophila FGFs have different activities and that cell migration and differentiation have different ligand requirements. Furthermore, these FGF ligands are not regulated solely by differential expression, but the sequences of these linked genes have evolved to serve different functions. We contend that inherent properties of FGF ligands make them suitable to support specific FGF-dependent processes, and that FGF ligands are not always interchangeable.

Graded Dorsal and Differential Gene Regulation in the Drosophila Embryo

A gradient of Dorsal activity patterns the dorsoventral (DV) axis of the early Drosophila melanogaster embryo by controlling the expression of genes that delineate presumptive mesoderm, neuroectoderm, and dorsal ectoderm. The availability of the Drosophila melanogaster genome sequence has accelerated the study of embryonic DV patterning, enabling the use of systems-level approaches. As a result, our understanding of Dorsal-dependent gene regulation has expanded to encompass a collection of more than 50 genes and 30 cis-regulatory sequences. This information, which has been integrated into a spatiotemporal atlas of gene regulatory interactions, comprises one of the best-understood networks controlling any developmental process to date. In this article, we focus on how Dorsal controls differential gene expression and how recent studies have expanded our understanding of Drosophila embryonic development from the cis-regulatory level to that controlling morphogenesis of the embryo.

Dynamic interpretation of hedgehog signaling in the Drosophila wing disc.

Drosophila cell response to the Hedgehog morphogen depends not just on a precise measurement of morphogen concentration at any given time, but instead on the history of cell exposure to morphogen.

Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

Dorsal-Ventral Gene Expression in the Drosophila Embryo Reflects the Dynamics and Precision of the Dorsal Nuclear Gradient

Patterning of the dorsal-ventral axis in the early Drosophila embryo depends on the nuclear distribution of the Dorsal transcription factor. Using live two-photon light-sheet microscopy, we quantified the nuclear Dorsal gradient in space and time and found that its amplitude and basal levels display oscillations throughout early embryonic development. These dynamics raise questions regarding how cells can reproducibly establish patterns of gene expression from a rapidly varying signal. We therefore quantified domains of Dorsal target genes, discovering their expression patterns are also dynamic. Computational modeling of this system reveals a correlation between Dorsal gradient dynamics and changes in target gene expression and suggests that these dynamics, together with time averaging of noise, results in the formation of graded gene expression borders in regions where the gradient is nearly flat. We propose that mRNA levels remain plastic during transient signaling events, allowing tissues to refine patterns in the face of genetic or environmental variation.

Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila

The NF-κB-related transcription factor, Dorsal, forms a nuclear concentration gradient in the early Drosophila embryo, patterning the dorsal-ventral (DV) axis to specify mesoderm, neurogenic ectoderm, and dorsal ectoderm cell fates. The concentration of nuclear Dorsal is thought to determine these patterning events; however, the levels of nuclear Dorsal have not been quantified previously. Furthermore, existing models of Dorsal-dependent germ layer specification and patterning consider steady-state levels of Dorsal relative to target gene expression patterns, yet both Dorsal gradient formation and gene expression are dynamic. We devised a quantitative imaging method to measure the Dorsal nuclear gradient while simultaneously examining Dorsal target gene expression along the DV axis. Unlike observations from other insects such as Tribolium, we find the Dorsal gradient maintains a constant bell-shaped distribution during embryogenesis. We also find that some classical Dorsal target genes are located outside the region of graded Dorsal nuclear localization, raising the question of whether these genes are direct Dorsal targets. Additionally, we show that Dorsal levels change in time during embryogenesis such that a steady state is not reached. These results suggest that the multiple gene expression outputs observed along the DV axis do not simply reflect a steady-state Dorsal nuclear gradient. Instead, we propose that the Dorsal gradient supplies positional information throughout nuclear cycles 10-14, providing additional evidence for the idea that compensatory combinatorial interactions between Dorsal and other factors effect differential gene expression along the DV axis.

Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity.

Migration is a complex, dynamic process that has largely been studied using qualitative or static approaches. As technology has improved, we can now take quantitative approaches towards understanding cell migration using in vivo imaging and tracking analyses. In this manner, we have established a four-step model of mesoderm migration during Drosophila gastrulation: (I) mesodermal tube formation, (II) collapse of the mesoderm, (III) dorsal migration and spreading and (IV) monolayer formation. Our data provide evidence that these steps are temporally distinct and that each might require different chemical inputs. To support this, we analyzed the role of fibroblast growth factor (FGF) signaling, in particular the function of two Drosophila FGF ligands, Pyramus and Thisbe, during mesoderm migration. We determined that FGF signaling through both ligands controls movements in the radial direction. Thisbe is required for the initial collapse of the mesoderm onto the ectoderm, whereas both Pyramus and Thisbe are required for monolayer formation. In addition, we uncovered that the GTPase Rap1 regulates radial movement of cells and localization of the beta-integrin subunit, Myospheroid, which is also required for monolayer formation. Our analyses suggest that distinct signals influence particular movements, as we found that FGF signaling is involved in controlling collapse and monolayer formation but not dorsal movement, whereas integrins are required to support monolayer formation only and not earlier movements. Our work demonstrates that complex cell migration is not necessarily a fluid process, but suggests instead that different types of movements are directed by distinct inputs in a stepwise manner.