Michelle T. Juarez

Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila.

The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. We have developed a variety of fluorescent reporters that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. We also find that four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-ß-cyclodextrin, and hydrogen peroxide) are sufficient to globally activate epidermal wound response genes in Drosophila embryos. We explore the epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals. Our results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating local repair and regeneration.

Multiple transcription factor codes activate epidermal wound–response genes in Drosophila

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes—Ddc, ple, msn, and kkv—that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound–response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view.

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.

Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues.

Toll pathway is required for wound-induced expression of barrier repair genes in the Drosophila epidermis

Significance After breaks in animal epidermal barriers, repair genes are activated in the cells adjacent to wound sites that help regenerate the barrier. The fruit fly Drosophila melanogaster is a favorable genetic system to find molecular detection systems that sense wounds and activate repair genes. In this paper, we find that the Toll signaling pathway, including the extracellular ligand Spätzle, the Toll receptor, and the Dif transcription factor, form a detection system to sense broken epidermis and then activate regeneration genes. The Toll pathway thus is involved not only in the activation of genes involved in fighting microbial invasion after epidermal breaks, but also in the activation of genes that regenerate epidermal barrier function. The epidermis serves as a protective barrier in animals. After epidermal injury, barrier repair requires activation of many wound response genes in epidermal cells surrounding wound sites. Two such genes in Drosophila encode the enzymes dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple). In this paper we explore the involvement of the Toll/NF-κB pathway in the localized activation of wound repair genes around epidermal breaks. Robust activation of wound-induced transcription from ple and Ddc requires Toll pathway components ranging from the extracellular ligand Spätzle to the Dif transcription factor. Epistasis experiments indicate a requirement for Spätzle ligand downstream of hydrogen peroxide and protease function, both of which are known activators of wound-induced transcription. The localized activation of Toll a few cell diameters from wound edges is reminiscent of local activation of Toll in early embryonic ventral hypoderm, consistent with the hypothesis that the dorsal–ventral patterning function of Toll arose from the evolutionary cooption of a morphogen-responsive function in wound repair. Furthermore, the combinatorial activity of Toll and other signaling pathways in activating epidermal barrier repair genes can help explain why developmental activation of the Toll, ERK, or JNK pathways alone fail to activate wound repair loci.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Communicating Science through a Novel Type of Journal

16:le2, 1 To the Editor: We would like to bring to the attention of the CBE—Life Sciences Education (LSE) community an online journal, Frontiers for Young Minds (http://kids.frontiersin.org), which is designed to promote scientific communication with the general public, especially young people. Launched in 2013 by a group from University of California at Berkeley led by Dr. Robert T. Knight, Frontiers for Young Minds serves as an openaccess resource that not only creates science literature for a younger audience but also brings kids into the review process. Specifically, scientists write kid-friendly versions of their articles, which are then reviewed by young people in the target age range for the pieces (ages 8–15). Authors “translate” the main ideas in the articles through the use of keywords as well as a glossary section to define any scientific nomenclature. Frontiers for Young Minds uses two article formats: 1) new discovery—to introduce a recent development in science by highlighting a previously published and peer-reviewed article; and 2) core concept—to provide a kid-friendly explanation of a fundamental scientific idea. A science mentor other than the authors guides the young reviewer through the review process. In an online discussion forum curated by the Frontiers for Young Minds editors, the authors and mentors discuss the comments from the young reviewers and work together to identify components of the articles that spark the curiosity of the kids and concepts that need further clarification. Frontiers for Young Minds publishes articles in a wide range of disciplines, including astronomy and space science, biodiversity, health, neuroscience, and earth and its resources. All articles include online profile links to all of the authors and the young reviewers. The submission process is free for the authors and follows an easy-to-navigate online platform to communicate with the editors, reviewers, and production staff. We published a new discovery format article in Frontiers for Young Minds as part of a creative writing project in Juarez’s research lab. We wrote a piece on a PLoS Genetics article (Juarez et al., 2011) on transcriptional responses to epidermal wounding in Drosophila (www.jove.com/video/50750/microinjection-wound-assay-vivo-localization -epidermal-wound-response). We also integrated content from an article published in Journal of Visualized Experiments (Juarez et al., 2013) to provide a visual aid for understanding how we conducted our wound assays. We created artistic interpretations of results and figures to illustrate key points of the research. For example, we created an “ouch scale” to quantify the results and help the young readers interpret them. We also created an image to highlight a curious child with a magnifying glass, a fruit fly, and DNA (Figure 1), because we hoped it would help young readers see themselves contributing to the scientific endeavor. The feedback from our young reviewer helped us to clarify the overall message of the article, especially as it related to fundamental concepts of DNA and genetics. The young reviewer was intrigued by the idea of doing experiments with Drosophila and using the results to impact human health. The final product of this writing exercise is a Frontiers for Young Minds article (http://kids .frontiersin.org/article/10.3389/frym.2016.00027). We invite LSE readers to share their biological research through Frontiers for Young Minds and to encourage their colleagues, including undergraduates, graduate students, postdoctoral associates, and other scientists, to do so. We found the process Michelle T. Juarez,* Chloe M. Kenet, and Chiandredi N. Johnson Sophie Davis Program in Biomedical Education, City University of New York School of Medicine, City College of New York, New York, NY 10031 Communicating Science through a Novel Type of Journal

Translating Research as an Approach to Enhance Science Engagement

The impact of research depends on the effective communication of discoveries. Scientific writing is the primary tool for the dissemination of research, and is an important skill that biomedical trainees have to develop. Despite its importance, scientific writing is not part of the mainstream curriculum. One strategy used to teach scientific writing is holding a journal club style discussion of primary research literature that the students are asked to read. However, this activity can result in a passive learning experience and limit the development of trainees’ scientific writing skills. In order to improve trainees’ written communication skills, we tested an exercise that involved generating a revised article describing prior research, in essence “translating” the science into basic language. Following the guidelines set out by “Frontiers for Young Minds” and feedback received from “Young Reviewers”, we wrote a revised article with a simpler description of the research. In this article, we describe this scientific writing exercise, which may ultimately serve as a model for scientists to share their research more efficiently in order to promote better public health outcomes.