Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging.

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Striped Expression of Leucine-Rich Repeat Proteins Coordinates Cell Intercalation and Compartment Boundary Formation in the Early Drosophila Embryo.

Planar polarity is a commonly observed phenomenon in which proteins display a consistent asymmetry in their subcellular localization or activity across the plane of a tissue. During animal development, planar polarity is a fundamental mechanism for coordinating the behaviors of groups of cells to achieve anisotropic tissue remodeling, growth, and organization. Therefore, a primary focus of developmental biology research has been to understand the molecular mechanisms underlying planar polarity in a variety of systems to identify conserved principles of tissue organization. In the early Drosophila embryo, the germband neuroectoderm epithelium rapidly doubles in length along the anterior-posterior axis through a process known as convergent extension (CE); it also becomes subdivided into tandem tissue compartments through the formation of compartment boundaries (CBs). Both processes are dependent on the planar polarity of proteins involved in cellular tension and adhesion. The enrichment of actomyosin-based tension and adherens junction-based adhesion at specific cell-cell contacts is required for coordinated cell intercalation, which drives CE, and the creation of highly stable cell-cell contacts at CBs. Recent studies have revealed a system for rapid cellular polarization triggered by the expression of leucine-rich-repeat (LRR) cell-surface proteins in striped patterns. In particular, the non-uniform expression of Toll-2, Toll-6, Toll-8, and Tartan generates local cellular asymmetries that allow cells to distinguish between cell-cell contacts oriented parallel or perpendicular to the anterior-posterior axis. In this review, we discuss (1) the biomechanical underpinnings of CE and CB formation, (2) how the initial symmetry-breaking events of anterior-posterior patterning culminate in planar polarity, and (3) recent advances in understanding the molecular mechanisms downstream of LRR receptors that lead to planar polarized tension and junctional adhesion.

Cellular, molecular, and biophysical control of epithelial cell intercalation.

Convergent extension is a conserved mechanism for elongating tissues. In the Drosophila embryo, convergent extension is driven by planar polarized cell intercalation and is a paradigm for understanding the cellular, molecular, and biophysical mechanisms that establish tissue structure. Studies of convergent extension in Drosophila have provided key insights into the force-generating molecules that promote convergent extension in epithelial tissues, as well as the global systems of spatial information that systematically organize these cell behaviors. A general framework has emerged in which asymmetrically localized proteins involved in cytoskeletal tension and cell adhesion direct oriented cell movements, and spatial signals provided by the Toll, Tartan, and Teneurin receptor families break planar symmetry to establish and coordinate planar cell polarity throughout the tissue. In this chapter, we describe the cellular, molecular, and biophysical mechanisms that regulate cell intercalation in the Drosophila embryo, and discuss how research in this system has revealed conserved biological principles that control the organization of multicellular tissues and animal body plans.

An LRR Receptor-Teneurin System Directs Planar Polarity at Compartment Boundaries.

Epithelial cells dynamically self-organize in response to extracellular spatial cues relayed by cell-surface receptors. During convergent extension in Drosophila, Toll-related receptors direct planar polarized cell rearrangements that elongate the head-to-tail axis. However, many cells establish polarity in the absence of Toll receptor activity, indicating the presence of additional spatial cues. Here we demonstrate that the leucine-rich-repeat receptor Tartan and the teneurin Ten-m provide critical polarity signals at epithelial compartment boundaries. The Tartan and Ten-m extracellular domains interact in vitro, and Tartan promotes Ten-m localization to compartment boundaries in vivo. We show that Tartan and Ten-m are necessary for the planar polarity and organization of compartment boundary cells. Moreover, ectopic stripes of Tartan and Ten-m are sufficient to induce myosin accumulation at stripe boundaries. These results demonstrate that the Tartan/Ten-m and Toll receptor systems together create a high-resolution network of spatial cues that guides cell behavior during convergent extension.

A positional Toll receptor code directs convergent extension in Drosophila.

Elongation of the head-to-tail body axis by convergent extension is a conserved developmental process throughout metazoans. In Drosophila, patterns of transcription factor expression provide spatial cues that induce systematically oriented cell movements and promote tissue elongation. However, the mechanisms by which patterned transcriptional inputs control cell polarity and behaviour have long been elusive. We demonstrate that three Toll family receptors, Toll-2, Toll-6 and Toll-8, are expressed in overlapping transverse stripes along the anterior-posterior axis and act in combination to direct planar polarity and polarized cell rearrangements during convergent extension. Simultaneous disruption of all three receptors strongly reduces actomyosin-driven junctional remodelling and axis elongation, and an ectopic stripe of Toll receptor expression is sufficient to induce planar polarized actomyosin contractility. These results demonstrate that tissue-level patterns of Toll receptor expression provide spatial signals that link positional information from the anterior-posterior patterning system to the essential cell behaviours that drive convergent extension.

The functions of grainy head-like proteins in animals and fungi and the evolution of apical extracellular barriers.

The Grainy head (GRH) family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi--organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl). We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2) mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins.

Three Drosophila Hox complex microRNAs do not have major effects on expression of evolutionarily conserved Hox gene targets during embryogenesis.

The discovery of microRNAs has resulted in a major expansion of the number of molecules known to be involved in gene regulation. Elucidating the functions of animal microRNAs has posed a significant challenge as their target interactions with messenger RNAs do not adhere to simple rules. Of the thousands of known animal microRNAs, relatively few microRNA:messenger RNA regulatory interactions have been biologically validated in an normal organismal context. Here we present evidence that three microRNAs from the Hox complex in Drosophila (miR-10-5p, miR-10-3p, miR-iab-4-5p) do not have significant effects during embryogenesis on the expression of Hox genes that contain high confidence microRNAs target sites in the 3' untranslated regions of their messenger RNAs. This is significant, in that it suggests that many predicted microRNA-target interactions may not be biologically relevant, or that the outcomes of these interactions may be so subtle that mutants may only show phenotypes in specific contexts, such as in environmental stress conditions, or in combinations with other microRNA mutations.

Silencing of an abdominal Hox gene during early development is correlated with limb development in a crustacean trunk.

We tested whether Artemia abd-A could repress limbs in Drosophila embryos, and found that although abd-A transcripts were produced, ABD-A protein was not. Similarly, developing Artemia epidermal cells showed expression of abd-A transcripts without accumulation of ABD-A protein. This finding in Artemia reveals a new variation in Hox gene function that is associated with morphological evolution. In this case, a HOX protein expression pattern is completely absent during early development, although the HOX protein is expressed at later stages in the central nervous system in a "homeotic-like" pattern. The combination of an absence of ABD-A protein expression in the Artemia limb primordia and the weak repressive function of Artemia UBX protein on the limb-promoting gene Dll are likely to be two reasons why homonomous limbs develop throughout the entire Artemia trunk.

Visualization of individual Scr mRNAs during Drosophila embryogenesis yields evidence for transcriptional bursting.

The detection and counting of transcripts within single cells via fluorescent in situ hybridization (FISH) has allowed researchers to ask quantitative questions about gene expression at the level of individual cells. This method is often preferable to quantitative RT-PCR, because it does not necessitate destruction of the cells being probed and maintains spatial information that may be of interest. Until now, studies using FISH at single-molecule resolution have only been rigorously carried out in isolated cells (e.g., yeast cells or mammalian cell culture). Here, we describe the detection and counting of transcripts within single cells of fixed, whole-mount Drosophila embryos via a combination of FISH, immunohistochemistry, and image segmentation. Our method takes advantage of inexpensive, long RNA probes detected with antibodies, and we present novel evidence to show that we can robustly detect single mRNA molecules. We use this method to characterize transcription at the endogenous locus of the Hox gene Sex combs reduced (Scr), by comparing a stably expressing group of cells to a group that only transiently expresses the gene. Our data provide evidence for transcriptional bursting, as well for divergent "accumulation" and "maintenance" phases of gene activity at the Scr locus.

Construction and characterization of deletions with defined end points in Drosophila using P elements in trans.

We used P-element transposase-mediated "male recombination" between two P elements in trans to create genetic deletions that removed a number of loci, including the gene encoding the neuropeptide crustacean cardioactive peptide (CCAP). Two classes of recombinant chromosomes were produced. Approximately one-quarter were viable when homozygous or hemizygous, whereas the remaining lines caused homozygous and hemizygous lethality. Preliminary analyses using PCR and CCAP immunohistochemistry suggested that, whereas the DNA of the viable lines was largely intact, most lethal lines contained chromosomal deletions that were roughly bounded by the insertion sites of the two P elements used. Southern blot analyses of select lethal lines showed that the DNA flanking the deletion was indeed grossly intact whereas the intervening DNA could not be detected. Sequencing across the deletion in three of these lethal lines identified a single line bearing intact genomic DNA on either side of the deletion separated by 30 bp of P-element DNA. The method described here suggests a simple procedure for creating deletions with defined end points. Importantly, it can use preexisting P-element insertion strains and does not rely on the use of transposable elements that are engineered to cause specific DNA rearrangements.

Site-specific transgenesis by Cre-mediated recombination in Drosophila.

Transposons such as P elements are routinely used to stably transfer exogenous DNA (transgenes) into the Drosophila genome. Transgene insertion events, however, are essentially random and are subject to 'position effects' from nearby endogenous regulatory elements. Here we describe a microinjection-based system that uses Cre-mediated recombination to insert transgenes into precise genomic 'landing sites'. The system is simple and efficient, and will permit precise comparisons between multiple transgenic constructs.

The role of binding site cluster strength in Bicoid-dependent patterning in Drosophila.

The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient that is critical for patterning the anterior-posterior (AP) body plan in Drosophila. Previous work identified several target genes that respond directly to Bcd-dependent activation. Positioning of these targets along the AP axis is thought to be controlled by cis-regulatory modules (CRMs) that contain clusters of Bcd-binding sites of different "strengths." Here we use a combination of Bcd-site cluster analysis and evolutionary conservation to predict Bcd-dependent CRMs. We tested 14 predicted CRMs by in vivo reporter gene assays; 11 show Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the most anterior part of the embryo, whereas others extend into middle and posterior regions. However, we do not detect a strong correlation between AP position of target gene expression and the strength of Bcd site clusters alone. Rather, we find that binding sites for other activators, including Hunchback and Caudal correlate with CRM expression in middle and posterior body regions. Also, many Bcd-dependent CRMs contain clusters of sites for the gap protein Kruppel, which may limit the posterior extent of activation by the Bcd gradient. We propose that the key design principle in AP patterning is the differential integration of positive and negative transcriptional information at the level of individual CRMs for each target gene.

A self-organizing system of repressor gradients establishes segmental complexity in Drosophila.

Gradients of regulatory factors are essential for establishing precise patterns of gene expression during development; however, it is not clear how patterning information in multiple gradients is integrated to generate complex body plans. Here we show that opposing gradients of two Drosophila transcriptional repressors, Hunchback (Hb) and Knirps (Kni), position several segments by differentially repressing two distinct regulatory regions (enhancers) of the pair-rule gene even-skipped (eve). Computational and in vivo analyses suggest that enhancer sensitivity to repression is controlled by the number and affinity of repressor-binding sites. Because the kni expression domain is positioned between two gradients of Hb, each enhancer directs expression of a pair of symmetrical stripes, one on each side of the kni domain. Thus, only two enhancers are required for the precise positioning of eight stripe borders (four stripes), or more than half of the whole eve pattern. Our results show that complex developmental expression patterns can be generated by simple repressor gradients. They also support the utility of computational analyses for defining and deciphering regulatory information contained in genomic DNA.