Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

A genome engineered tool set for Drosophila TGF-ß/BMP signaling studies.

Ligands of the TGF-ß/BMP superfamily are critically involved in the regulation of growth, patterning and organogenesis and can act as long-range morphogens. Essential for understanding TGF-ß/BMP signaling dynamics and regulation are tools that allow monitoring and manipulating pathway components expressed at physiological levels and endogenous spatiotemporal patterns. We used genome engineering to generate a comprehensive library of endogenously epitope- or fluorescently-tagged versions of receptors, co-receptors, transcription factors and key feedback regulators of the Drosophila BMP and Activin signaling pathways. We demonstrate that the generated alleles are biologically active and can be utilized for assessing tissue and subcellular distribution of the corresponding proteins. Further, we show that the genomic platforms can be used for in locus structure-function and cis-regulatory analyses. Finally, we present a complementary set of protein binder-based tools, which allow visualization as well as manipulation of the stability and subcellular localization of epitope-tagged proteins, providing new tools for the analysis of BMP signaling and beyond.

Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex.

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are misregulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex, which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high-throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate.

Antennapedia: The complexity of a master developmental transcription factor.

Hox genes encode transcription factors that play an important role in establishing the basic body plan of animals. In Drosophila, Antennapedia is one of the five genes that make up the Antennapedia complex (ANT-C). Antennapedia determines the identity of the second thoracic segment, known as the mesothorax. Misexpression of Antennapedia at different developmental stages changes the identity of the mesothorax, including the muscles, nervous system, and cuticle. In Drosophila, Antennapedia has two distinct promoters highly regulated throughout development by several transcription factors. Antennapedia proteins are found with other transcription factors in different ANTENNAPEDIA transcriptional complexes to regulate multiple subsets of target genes. In this review, we describe the different mechanisms that regulate the expression and function of Antennapedia and the role of this Hox gene in the development of Drosophila.

Dilp8 and its candidate receptor, Drl, are involved in the transdetermination of the Drosophila imaginal disc.

Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. We previously showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, we found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. We propose a model in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD.

Hherisomes, Hedgehog specialized recycling endosomes, are required for high level Hedgehog signaling and tissue growth.

In metazoans, tissue growth and patterning is partly controlled by the Hedgehog (Hh) morphogen. Using immuno-electron microscopy on Drosophila wing imaginal discs, we identified a cellular structure, the Hherisomes, which contain the majority of intracellular Hh. Hherisomes are recycling tubular endosomes, and their formation is specifically boosted by overexpression of Hh. Expression of Rab11, a small GTPase involved in recycling endosomes, boosts the size of Hherisomes and their Hh concentration. Conversely, increased expression of the transporter Dispatched, a regulator of Hh secretion, leads to their clearance. We show that increasing Hh density in Hherisomes through Rab11 overexpression enhances both the level of Hh signaling and disc pouch growth, whereas Dispatched overexpression decreases high-level Hh signaling and growth. We propose that, upon secretion, a pool of Hh triggers low-level signaling, whereas a second pool of Hh is endocytosed and recycled through Hherisomes to stimulate high-level signaling and disc pouch growth. Altogether, our data indicate that Hherisomes are required to sustain physiological Hh activity necessary for patterning and tissue growth in the wing disc.

Model systems for regeneration: Drosophila.

Drosophila melanogaster has historically been a workhorse model organism for studying developmental biology. In addition, Drosophila is an excellent model for studying how damaged tissues and organs can regenerate. Recently, new precision approaches that enable both highly targeted injury and genetic manipulation have accelerated progress in this field. Here, we highlight these techniques and review examples of recently discovered mechanisms that regulate regeneration in Drosophila larval and adult tissues. We also discuss how, by applying these powerful approaches, studies of Drosophila can continue to guide the future of regeneration research.

Establishing compartment boundaries in Drosophila wing imaginal discs: An interplay between selector genes, signaling pathways and cell mechanics.

The partitioning of cells into groups or 'compartments' separated by straight and sharp boundaries is important for tissue formation in animal development. Cells from neighboring compartments are characterized by distinct fates and functions and their continuous separation at compartment boundaries maintains proper tissue organization. Signaling across compartment boundaries can induce the local expression of morphogens that in turn direct growth and patterning of the surrounding cells. Compartment boundaries play therefore an important role in tissue development. Compartment boundaries were first identified in the early 1970s in the Drosophila wing. Here, we review the role of compartment boundaries in growth and patterning of the developing wing and then discuss the genetic and physical mechanisms underlying cell separation at compartment boundaries in this tissue.

Single-Cell Atlas of the Drosophila Leg Disc Identifies a Long Non-Coding RNA in Late Development.

The Drosophila imaginal disc has been an excellent model for the study of developmental gene regulation. In particular, long non-coding RNAs (lncRNAs) have gained widespread attention in recent years due to their important role in gene regulation. Their specific spatiotemporal expressions further support their role in developmental processes and diseases. In this study, we explored the role of a novel lncRNA in Drosophila leg development by dissecting and dissociating w1118 third-instar larval third leg (L3) discs into single cells and single nuclei, and performing single-cell RNA-sequencing (scRNA-seq) and single-cell assays for transposase-accessible chromatin (scATAC-seq). Single-cell transcriptomics analysis of the L3 discs across three developmental timepoints revealed different cell types and identified lncRNA:CR33938 as a distal specific gene with high expression in late development. This was further validated by fluorescence in-situ hybridization (FISH). The scATAC-seq results reproduced the single-cell transcriptomics landscape and elucidated the distal cell functions at different timepoints. Furthermore, overexpression of lncRNA:CR33938 in the S2 cell line increased the expression of leg development genes, further elucidating its potential role in development.

Nano-CT imaging of larvae in the ant Pheidole hyatti reveals coordinated growth of a rudimentary organ necessary for soldier development.

The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes-the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small-headed minor workers and larger, big-headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head-to-body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano-CT imaging and three-dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye-antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye-antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter-organ coordination as compared with other insects to generate the big-headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter-organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution.

Fat regulates expression of four-jointed reporters in vivo through a 20 bp element independently of the Hippo pathway.

Development of an organism requires accurate coordination between the growth of a tissue and orientation of cells within the tissue. The large cadherin Fat has been shown to play a role in both of these processes. Fat is involved in the establishment of planar cell polarity and regulates growth through the Hippo pathway, a developmental cascade that controls proliferation and apoptosis. Both Fat and the Hippo pathway are known to regulate transcription of four-jointed, although the nature of this regulation is unknown. In this study, we test whether Fat affects four-jointed transcription via or independently of Hippo pathway. Our analysis of the four-jointed regulatory region reveals a 1.2 kb element that functions as an enhancer for graded expression of Four-jointed in the eye imaginal disc. Within this enhancer element, we identify a 20 bp fragment that is critical for regulation by Fat but not by Hippo. Our findings suggest that Fat and the Hippo pathway control four-jointed expression independently of each other and none of the transcription factors known to function downstream of the Hippo pathway are required to regulate four-jointed expression through the 1.2 kb element.

Cell dynamics underlying oriented growth of the Drosophila wing imaginal disc.

Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.

Proximal fate marker homothorax marks the lateral extension of stalk-eyed fly Cyrtodopsis whitei.

The placement of eyes on insect head is an important evolutionary trait. The stalk-eyed fly, Cyrtodopsis whitei, exhibits a hypercephaly phenotype where compound eyes are located on lateral extension from the head while the antennal segments are placed inwardly on this stalk. This stalk-eyed phenotype is characteristic of the family Diopsidae in the Diptera order and dramatically deviates from other dipterans, such as Drosophila. Like other insects, the adult eye and antenna of stalk-eyed fly develop from a complex eye-antennal imaginal disc. We analyzed the markers involved in proximo-distal (PD) axis of the developing eye imaginal disc of the stalk-eyed flies. We used homothorax (hth) and distalless (dll), two highly conserved genes as the marker for proximal and distal fate, respectively. We found that lateral extensions between eye and antennal field of the stalk-eyed fly's eye-antennal imaginal disc exhibit robust Hth expression. Hth marks the head specific fate in the eye- and proximal fate in the antenna-disc. Thus, the proximal fate marker Hth expression evolves in the stalk-eyed flies to generate lateral extensions for the placement of the eye on the head. Moreover, during pupal eye metamorphosis, the lateral extension folds back on itself to place the antenna inside and the adult compound eye on the distal tip. Interestingly, the compound eye in other insects does not have a prominent PD axis as observed in the stalk-eyed fly.

Nuclear Lamin is required for Winged Eye-mediated transdetermination of Drosophila imaginal disc.

Drosophila imaginal discs often change their cell fate under stress conditions, and this phenomenon, called transdetermination (TD), has long been a useful model for studying cell fate plasticity during regeneration. We previously identified a chromatin-associated protein, Winged Eye (Wge), which induces eye-to-wing TD upon its over-expression in eye imaginal discs. However, the molecular mechanism of Wge-mediated TD remains obscure. Here, we analyzed Wge-interacting proteins and found that several heterochromatin-related proteins, including a nuclear lamina protein, Lamin (Lam), were associated with Wge protein in cultured cells. Knockdown experiments revealed that Lam is indeed required for Wge-mediated eye-to-wing TD. Moreover, Wge over-expression altered the spatial organization of genomic DNA inside the cell nuclei. Accordingly, we suggest that Wge interacts with Lam to link some genomic regions with the nuclear periphery and regulates chromatin dynamics in imaginal disc TD.

The fly eye: Through the looking glass.

The developing eye-antennal disc of Drosophila melanogaster has been studied for more than a century, and it has been used as a model system to study diverse processes, such as tissue specification, organ growth, programmed cell death, compartment boundaries, pattern formation, cell fate specification, and planar cell polarity. The findings that have come out of these studies have informed our understanding of basic developmental processes as well as human disease. For example, the isolation of a white-eyed fly ultimately led to a greater appreciation of the role that sex chromosomes play in development, sex determination, and sex linked genetic disorders. Similarly, the discovery of the Sevenless receptor tyrosine kinase pathway not only revealed how the fate of the R7 photoreceptor is selected but it also helped our understanding of how disruptions in similar biochemical pathways result in tumorigenesis and cancer onset. In this article, I will discuss some underappreciated areas of fly eye development that are fertile for investigation and are ripe for producing exciting new breakthroughs. The topics covered here include organ shape, growth control, inductive signaling, and right-left symmetry. Developmental Dynamics 247:111-123, 2018. © 2017 Wiley Periodicals, Inc.

Approximate Bayesian computation reveals the importance of repeated measurements for parameterising cell-based models of growing tissues.

The growth and dynamics of epithelial tissues govern many morphogenetic processes in embryonic development. A recent quantitative transition in data acquisition, facilitated by advances in genetic and live-imaging techniques, is paving the way for new insights to these processes. Computational models can help us understand and interpret observations, and then make predictions for future experiments that can distinguish between hypothesised mechanisms. Increasingly, cell-based modelling approaches such as vertex models are being used to help understand the mechanics underlying epithelial morphogenesis. These models typically seek to reproduce qualitative phenomena, such as cell sorting or tissue buckling. However, it remains unclear to what extent quantitative data can be used to constrain these models so that they can then be used to make quantitative, experimentally testable predictions. To address this issue, we perform an in silico study to investigate whether vertex model parameters can be inferred from imaging data, and explore methods to quantify the uncertainty of such estimates. Our approach requires the use of summary statistics to estimate parameters. Here, we focus on summary statistics of cellular packing and of laser ablation experiments, as are commonly reported from imaging studies. We find that including data from repeated experiments is necessary to generate reliable parameter estimates that can facilitate quantitative model predictions.

The Imaginal Disc Growth Factors 2 and 3 participate in the Drosophila response to nematode infection.

The Drosophila imaginal disc growth factors (IDGFs) induce the proliferation of imaginal disc cells and terminate cell proliferation at the end of larval development. However, the participation of Idgf-encoding genes in other physiological processes of Drosophila including the immune response to infection is not fully understood. Here, we show the contribution of Idgf2 and Idgf3 in the Drosophila response to infection with Steinernema carpocapsae nematodes carrying or lacking their mutualistic Xenorhabdus nematophila bacteria (symbiotic or axenic nematodes, respectively). We find that Idgf2 and Idgf3 are upregulated in Drosophila larvae infected with symbiotic or axenic Steinernema and inactivation of Idgf2 confers a survival advantage to Drosophila larvae against axenic nematodes. Inactivation of Idgf2 induces the Imd and Jak/Stat pathways, whereas inactivation of Idgf3 induces the Imd, Toll and Jak/Stat pathways. We also show that inactivation of the Imd pathway receptor PGRP-LE upregulates Idgf2 against Steinernema nematode infection. Finally, we demonstrate that inactivation of Idgf3 induces the recruitment of larval haemocytes in response to Steinernema. Our results indicate that Idgf2 and Idgf3 might be involved in different yet crucial immune functions in the Drosophila antinematode immune response. Similar findings will promote the development of new targets for species-specific pest control strategies.

RhoGAP68F controls transport of adhesion proteins in Rab4 endosomes to modulate epithelial morphogenesis of Drosophila leg discs.

Elongation and invagination of epithelial tissues are fundamental developmental processes that contribute to the morphogenesis of embryonic and adult structures and are dependent on coordinated remodeling of cell-cell contacts. The morphogenesis of Drosophila leg imaginal discs depends on extensive remodeling of cell contacts and thus provides a useful system with which to investigate the underlying mechanisms. The small Rho GTPase regulator RhoGAP68F has been previously implicated in leg morphogenesis. It consists of on an N-terminal Sec14 domain and a C-terminal GAP domain. Here we examined the molecular function and role of RhoGAP68F in epithelial remodeling. We find that depletion of RhoGAP68F impairs epithelial remodeling from a pseudostratified to simple, while overexpression of RhoGAP68F causes tears of lateral cell-cell contacts and thus impairs epithelial integrity. We show that the RhoGAP68F protein localizes to Rab4 recycling endosomes and forms a complex with the Rab4 protein. The Sec14 domain is sufficient for localizing to Rab4 endosomes, while the activity of the GAP domain is dispensable. RhoGAP68F, in turn, inhibits the scission and movement of Rab4 endosomes involved in transport the adhesion proteins Fasciclin3 and E-cadherin back to cell-cell contacts. Expression of RhoGAP68F is upregulated during prepupal development suggesting that RhoGAP68F decreases the transport of key adhesion proteins to the cell surface during this developmental stage to decrease the strength of adhesive cell-cell contacts and thereby facilitate epithelial remodeling and leg morphogenesis.

TIE-DYE: a combinatorial marking system to visualize and genetically manipulate clones during development in Drosophila melanogaster.

Two types of information are particularly valuable in understanding the development of a tissue or an organ from a small population of founder cells. First, it is useful to know the composition of the final structure in terms the contribution of individual founder cells. Second, it is important to understand cell-cell interactions. To facilitate the study of both of these aspects of organ development at a tissue-wide level, we have developed a method, TIE-DYE, that allows simultaneous lineage tracing of multiple cell populations as well as the genetic manipulation of a subset of these populations. Seven uniquely marked categories of cells are produced by site-directed recombination of three independent cassettes. We have used the TIE-DYE method to estimate the number of founder cells that give rise to the wing-imaginal disc during normal development and following compensatory growth caused by X-ray irradiation of the founder cells. We also show that four out of the seven types of marked clones can be genetically manipulated by gene overexpression or RNAi knockdown, allowing an assessment of the consequences of these manipulations on the entire wing disc. We demonstrate the utility of this system in studying the consequences of alterations in growth, patterning and cell-cell affinity.

A global pattern of mechanical stress polarizes cell divisions and cell shape in the growing Drosophila wing disc.

Organismal development is under genetic control. Ultimately, mechanical forces shape embryos. If we want to understand the precise regulation of size and shape in animals, we must dissect how forces are distributed in developing tissues, and how they drive cell behavior to shape organs. This has not been addressed fully in the context of growing tissues. As cells grow and divide, they exert a pressure on their neighbors. How these local stresses add up or dissipate as the tissue grows is an unanswered question. We address this issue in the growing wing imaginal disc of Drosophila larvae, the precursor of the adult wing. We used a quantitative approach to analyze the strains and stresses of cells of the wing pouch, and found a global pattern of stress whereby cells in the periphery of the tissue are mechanically stretched and cells in the center are compressed. This pattern has important consequences on cell shape in the wing pouch: cells respond to it by polarizing their acto-myosin cortex, and aligning their divisions with the main axis of cell stretch, thereby polarizing tissue growth. Ectopic perturbations of tissue growth by the Hippo signaling pathway reorganize this pattern in a non-autonomous manner, suggesting a synergy between tissue mechanics and growth control during wing disc morphogenesis.