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

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.