Biology Open

Cells and tissues have the ability to respond to different stresses and maintain their homeostasis. The b-Zip transcription factor Xrp1 was found to play a pivotal role in this context in Drosophila. Xrp1 is required for the response to irradiation, in genetic conditions of ribosomal protein (Rp) deficiency leading to cell competition, and more generally in mediating the Integrated Stress Response (ISR). However, how these diverse stresses converge to and activate Xrp1 is still unclear. Our results demonstrate the existence of three separate mechanisms of Xrp1 induction. In response to Rp stress, a short isoform of Xrp1 is produced and requires the function of the ribosomal protein RpS12. In response to irradiation, a specific mRNA isoform is transcriptionally induced, which encodes the Xrp1-short protein isoform, in a p53-dependent manner. In response to the ISR, the long isoform of Xrp1 is translated by the derepression of a uORF present on the Xrp1 5'UTR, a mechanism that requires eIF2 phosphorylation, RpS12, and a transcriptional induction by ATF4. Therefore, our work provides a comprehensive understanding of the role of Xrp1 in different situations of cellular stress, illustrating the complex level of integration of the mechanisms controlling this central gene.

Epithelial tube morphogenesis is critical for the function of many organs. Basement membranes underlie epithelia and their remodeling is a key step to reach the correct size and shape. Key regulators that mediate basement membrane remodeling for tube elongation and branching remain largely unknown. We analyze the expression and function of AdamTS-B, a matrix metalloprotease, in the respiratory system of Drosophila. Here we show, that AdamTS-B is expressed early in tracheal development during placode formation. We generated a mutant line of AdamTS-B, which is lethal. Analysis of trachea morphogenesis in this AdamTS-B mutant reveal a function in tube elongation and cell migration. Our results suggest that AdamTS-B control BM remodeling required for organ shape.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

The tunicate Ciona robusta offers a tractable non-vertebrate chordate model for probing gene function via tissue-specific, CRISPR/Cas9-mediated mutagenesis in F0. Building on Arcadia Sciences Zoogle platform, which identifies and ranks orthologs of human genes from various non-traditional model organisms, we carried out a pilot project to probe the developmental roles of three notochord- and endoderm-expressed candidate orthologs of human disease genes (Fcho, Pgm3, and Nckap1) alongside a fourth gene (Plastin) implicated in papilla cell elongation. This preprint compiles and updates a series of research project milestones previously posted episodically on Zenodo. Here we summarize the full results and our conclusion about this pilot project. Using CRISPR/Cas9, we found that tissue-specific knockout of Pgm3 and, to a lesser extent, Fcho caused significant defects in larval tail elongation. Separately, CRISPR knockout of Plastin, an actin-bundling gene expressed throughout the sensory-adhesive papillae of the larva, caused a subtle reduction in papilla cell elongation when combined as a duoble knockout with another actin-bundling protein-encoding gene, Villin. These results identify Pgm3 as the most promising candidate for further development as a Ciona-based model of human disease and demonstrate the utility of tissue-specific CRISPR screening for prioritizing candidate disease gene orthologs identified through comparative genomics platforms like Zoogle.

Adult stem cells can survive diverse insults, from ionizing radiation to toxic chemicals. In vertebrates, ABC transporter expression and dye-efflux capacity have been associated with stem-cell protection and multidrug resistance. How compound accumulation differs between adult stem cells and their differentiated daughters within an intact tissue remains poorly understood. Here, we show that adult Drosophila intestinal stem/progenitor cells maintain a distinct low-accumulation state for membrane-permeable compounds and that ABC transporters contribute to this phenotype and to the response to cytotoxic stress. Across nine membrane-permeable probes, stem/progenitor cells accumulated eight at lower levels than differentiated enterocytes. We selected tetramethylrhodamine methyl ester (TMRM), a membrane-permeable fluorescent probe that showed a strong difference in accumulation between stem/progenitor cells and enterocytes, as a quantitative readout for an RNAi screen of all 56 Drosophila ABC-family genes. The screen identified seven candidate transporters whose depletion increased the stem/progenitor-to-enterocyte TMRM fluorescence ratio. Further characterization of one of them, ABCB7, showed that ABCB7-depleted guts contained fewer mitotic cells during cytotoxic drug exposure. These results implicate ABC transporters in lower compound accumulation in Drosophila intestinal stem/progenitor cells and support a role for ABC transporters in their response to cytotoxic challenge.

PAX9 is an evolutionarily conserved paired-box transcription factor that is critical for embryonic development and human diseases. The mouse model has been predominantly used to investigate Pax9 functions. Zebrafish has emerged as a complementary vertebrate model for various human diseases, including cancers. Until recently, the functions of the zebrafish pax9 gene in jaw and hematopoiesis have started to be uncovered. However, detailed pax9 spatiotemporal expression, molecular mechanisms, and potential functions in other zebrafish organs remain largely unknown. With the technical advances in CRISPR-Cas9, non-homologous end joining (NHEJ) has made knockin and knockout a convenient way to examine endogenous gene expression in vivo and to generate a loss-of-function allele simultaneously. Here, we first generated pax9 knockin fish lines by inserting fluorescent proteins at the start of the endogenous pax9 coding region. Then, we examined pax9 expression in real time from early embryonic stages through adulthood. Except for previously reported expression domains, we were able to identify pax9 expression in high resolution in the paired and median fins, where pax9 marks anterior fin rays. Moreover, our knockin and knockout mutants showed increased fin ray number in median fins, but no evident effect on paired fins in pax9 null mutants. Thus, PAX9 is critical for median fin patterning in zebrafish.

Stable intercellular bridges that form through incomplete cytokinesis are present in a wide variety of cell types but their function in somatic cell differentiation is not well understood. Using super-resolution confocal microscopy, we identified a stepwise process of ring canal development in the follicle cells of the Drosophila ovary. Using a custom-trained deep learning model to aid in 3D image segmentation and follicle cell quantification, we found that ring canals are more heterogeneous at the early prefollicle stages compared to the more differentiated later stages. In addition, we found that depletion of the septin, peanut, caused cytokinetic defects but did not disrupt ring canal formation, whereas depletion of the ESCRT III gene, shrub, increased ring canal number in prefollicle cells and caused proliferation and differentiation phenotypes. Our findings identify cytokinesis and ring canal formation as a point of regulation in the patterning of proliferation and differentiation in the Drosophila follicle stem cell lineage.

Hox genes play central roles in specifying positional identities along the vertebrate anterior-posterior axis. In mice, genetic analyses have demonstrated that Hox genes distributed among the four Hox clusters contribute to vertebral patterning, with extensive functional redundancy among paralogous genes. Our previous genetic analysis in zebrafish identified important roles for HoxB- and HoxC-related genes in specifying anterior vertebral identities, whereas the contributions of HoxA- and HoxD-related genes remained unresolved. Here, we examined adult zebrafish carrying extensive combinations of hoxaa, hoxab, and hoxda cluster deletions and generated five-gene homozygous mutants carrying frameshift mutations in hoxa3a, hoxa4a, hoxa5a, hoxd3a, and hoxd4a. X-ray micro-CT analysis revealed no obvious alterations in anterior vertebral morphology in either the compound cluster mutants or the five-gene mutants. These results indicate that HoxA/D genes make only a limited detectable contribution to anterior vertebral patterning in zebrafish. Together with our previous findings, they suggest that vertebral patterning functions are distributed unevenly among zebrafish Hox clusters, with a predominant contribution from HoxB/C-related genes.

Transfection of cells with DNA plasmids typically involves the uptake of lipoparticles by endocytosis, followed by the inefficient escape of these particles from endosomes into the cytoplasm. We found that the expression of transfected plasmids was reduced in cells depleted of either SEPTIN 9 or proteins in the endosomal sorting complexes required for transport (ESCRT) pathway. The reduction in plasmid expression could not be fully explained by effects on endocytosis. SEPTIN 9 depletion appeared to reduce the acidification of plasmid-containing compartments, suggesting that it primarily affects the pH-sensitive escape of plasmids from endosomes. Depletion of the ESCRT proteins VPS36 or ALIX resulted in especially dramatic reductions in transfected plasmid expression, which were accompanied by reduced colocalization between the transfected DNA and CHMP4, an ESCRT protein important for endosomal membrane remodeling during intraluminal vesicle formation. Finally, transfected plasmid DNA was strongly colocalized with LC3B, suggesting that the default pathway for transfected material is autophagy.

Background: In humans, haploinsufficiency of TWIST1 leads to Saethre-Chotzen Syndrome, characterized by limb abnormalities and craniofacial defects. In mouse, Twist1 promotes limb bud outgrowth via maintenance of the apical ectodermal ridge (AER), in addition to its craniofacial roles. In zebrafish, two twist1 paralogs, twist1a and twist1b, are known to influence cranial neural crest and cranial suture development but have not yet been implicated in limb formation. Results: We find that twist1a and twist1b act redundantly during zebrafish pectoral fin development. While twist1b mutants have normal fins, twist1a mutants have small fins, and twist1a;twist1b double mutants lack fins. These defects appear to originate with impaired establishment of the pectoral fin field, followed by failure of AER formation, likely due to reduced fgf10a expression in the fin mesenchyme. Anterior-posterior patterning of the fin bud is relatively normal in twist1a mutants and twist1a;twist1b double mutants, albeit with an anterior extension of shha expression in some embryos. Conclusions: Together, our results show that twist1a and twist1b play crucial and overlapping roles in zebrafish pectoral fin development, echoing roles played by Twist1 in the mouse limb bud and highlighting an early impact of twist1 genes on the initial formation of the zebrafish forelimb field.

Notch signaling is essential for maintaining intestinal stem cell activity and directing epithelial cell fate. Paneth cells have been proposed to be niche cells, providing Notch signal to neighboring stem cells through expression of the key Notch ligands DLL1 and DLL4. However, intestinal stem cells persist in the absence of Paneth cells. Here, we used genetic mouse models to clarify the role of Paneth cell-derived Notch ligands for stem cell function. Paneth cell-specific deletion of Dll1 and Dll4 resulted in loss of crypt base stem cells, with no effect on overall crypt cell proliferation. Organoid growth was reduced in knockout mice, suggesting reduced stem cell function. Upon irradiation injury, stem cell return and crypt regeneration were significantly impaired in the Notch ligand-deleted mice. These findings suggest that Paneth cell-derived Notch ligands DLL1 and DLL4 are crucial for crypt base stem cell maintenance and for crypt regeneration after injury.

Primordial germ cells (PGCs) are the stem cell lineage responsible for transmitting genetic information across generations and are therefore central to studies of germline development, reproduction, and evolution. However, a lack of molecular tools in jawless vertebrates such as lampreys and hagfish has limited comparative insights into the evolutionary origins of vertebrate germline cells. Here, we report the development of the first germline-specific reporter (GSR) in the sea lamprey (Petromyzon marinus), Tg(piwil1: egfp-UTRnanos1), a piwil1-based germline reporter that is driven by the sea lamprey piwil1 regulatory region that directs Green Fluorescent Protein (GFP) expression in early PGCs. Promoter activity was validated by robust GFP expression in PGCs of zebrafish embryos co-injected with Tol2 transposase mRNA. Injection of the construct into sea lamprey embryos resulted in early, persistent, and gonad-localized GFP-positive cells. These results demonstrate stable germline-specific transgene expression in a jawless vertebrate and suggest deep evolutionary conservation of germline regulatory mechanisms between jawless and jawed vertebrates. Furthermore, the piwil1-based germline reporter line enabled lifetime visualization of zebrafish germline cells, revealing that increased PGC abundance during embryogenesis biases toward female sexual differentiation. Together, this study establishes the first GSR system in a jawless vertebrate and provides a useful platform for investigating germline biology and vertebrate germline evolution.

During neurotransmission, synaptic vesicle exocytosis adds membrane and proteins to the cell surface. To sustain further release, this material must be retrieved, via several distinct endocytic modes matched to the level of exocytosis. The GTPase dynamin plays a central role in endocytosis, but it has remained unclear which endocytic modes it supports. In mammals, distinct dynamin gene products with different proline-rich domains (PRDs) are proposed to mediate particular modes of endocytosis; however, the function of each PRD isoform has not been tested in an organism. Drosophila dynamin is encoded by one gene (shibire) that produces long and short PRD isoforms (Shi-L and Shi-S), which differ by a 48 amino acid C-terminal extension. Using isoform-specific knockin and knockdown tools, we found that loss of the more abundant Shi-S isoform disrupted bulk endocytosis and vesicle reformation under high exocytic demand, reduced evoked transmission at moderate levels of activity, and enhanced spontaneous release at rest. These functions did not depend on the PRD extension, as either isoform could rescue these phenotypes when re-expressed. Our results indicate that dynamin contributes to vesicle recycling across multiple endocytic retrieval modes and that PRD specialization is not required for these functions.

Site-specific recombinases, such as Cre recombinase (Cre) and FLP recombinase (FLPase), are popular tools that enable precise spatiotemporal control over fluorescent reporter genes. However, studies show that increased Cre expression can lead to cytotoxic phenotypes including DNA damage, cell cycle arrest, cell death, and aneuploidy. In contrast, there is little-to-no known cytotoxicity associated with FLPase. FLPase is considered to be the safer, if less efficient, recombinase. To compensate for decreased efficiency, researchers will often increase expression of the FLPase to ensure a complete gene edit. Despite previous evidence that FLPase is nontoxic at high doses, we report that high expression of FLPase from multi-copy extrachromosomal arrays can disrupt synaptic protein trafficking in C. elegans. We discovered several off-target effects of high FLPase expression including mislocalization of the pre-synaptic marker FLPon::mScarlet::SYD-2 and decreased synaptic abundance, transport, and expression of the ionotropic glutamate receptor subunit GLR-1. Neither overexpression of an inactive FLPase, low activity FLPase, nor mCherry under the same promoter elicited these effects. However, single-copy expression of the FLPase construct reliably tags FLPon::mScarlet::SYD-2 and does not lead to these off-target effects. This work supports growing awareness that tyrosine recombinases can become cytotoxic at high doses.

Abstract The canonical WNT signaling pathway guides cell growth throughout the human lifespan. In adults, WNT plays an essential role in tissue homeostasis by maintaining tissue stem cells. In the intestine, the WNT transcription factor, TCF4, is necessary for stem cell maintenance. Aberrant activation of WNT signaling is generally accepted as an initiating event in colon cancer, leading to constitutive TCF4 activity. To investigate the role of this key transcription factor in global gene regulation, we abrogated TCF4 expression in colon cancer cells. Loss of TCF4 function resulted in the over-expression of the WNT transcription factor, LEF1. LEF1 was transcriptionally competent and over-compensated for TCF4 (TCF7L2) silencing in a WNT reporter assay. TCF4 enlisted the transcriptional repressor CtBP1, and bound the LEF1 promoter, indicative of direct repression. TCF4 and LEF1 drove different transcriptional programs, with TCF4 favoring MYC and cell cycle progression, while LEF1 favored immune-related gene signatures. TCF4 was the primary regulator of MYC expression, yet also the mediator of LEF1 repression, suggestive of competition centered on TCF4 transcriptional output despite the {beta}-catenin saturated colon cancer nucleus. We conclude that TCF4 has dual activator-repressor activity in colon cancer.

Background: The insect cuticle is a chitin-containing extracellular matrix whose formation requires precise coordination of chitin biosynthesis, fibril organization, and post-synthetic maturation. Although lipids are essential components of the cuticle, the molecular mechanisms linking lipid transport to chitin deposition remain poorly understood. Here we identify TcFabp8, a fatty acid-binding protein in Tribolium castaneum, as a functionally important regulator of chitin synthase 1 (Chs1)-dependent cuticle formation. Results: RNAi-mediated depletion of TcFABP8 caused molting defects, reduced cuticle thickness, loss of apical TcChs1 immunoreactivity, and reduced chitin deposition and organization. The finding that TcCHS1 transcript levels were unaffected by RNAi targeting TcFABP8 suggests that a post-transcriptional step, potentially involving vesicular TcChs1 transport, is affected, thereby impairing chitin synthesis and deposition. Synchrotron X-ray diffraction analysis of adult elytra revealed that TcFabp8 depletion preserved characteristic -chitin-associated scattering features, but altered chitin fibril accumulation, crystalline coherence, and the spatially ordered alignment of fibrils within the cuticular matrix. The TcFabp8 gene locus encodes splice variants yielding different N-terminal architectures: TcFabp8X1 carries a Sec/SPI signal peptide which is absent in TcFabp8X2 and TcFabp8X3. Immunocytochemistry using TcFabp8-specific antibodies detected intracellular signals in subapical cytoplasmic puncta and extracellular signals within the cuticle layers, which partially colocalized with Nile red fluorescence indicating lipid-rich environments. Conclusions: Together, our data demonstrate that TcFabp8 is a critical facilitator of TcChs1-dependent chitin deposition and extends functional connection between lipid transport and chitin biosynthesis in insects.

Butterfly wings present a tremendous gallery of colorful patterns, offering a unique opportunity to study how developmental pattern formation processes evolve. We still do not understand the genetic basis of several key aspects of wing pattern development, however. Three paralogous POU domain transcription factors nubbin, ventral veinless (vvl), and pdm3 are all known wing development genes in Drosophila melanogaster. Here we combine gene expression and knockout approaches to show that each of these genes plays multiple novel wing patterning roles in the common buckeye butterfly, Junonia coenia. We found that nubbin controls eyespot pattern determination via a non-cell autonomous repressor-like effect originating at the wing veins, such that nubbin knockouts have larger eyespots. nubbin also regulates pigment identity and scale morphology across the wings. We also found that vvl regulates pigment identity of the discal bands and ventral hindwing. Last, we found that pdm3 is required for determining the outer rings of eyespot patterns, where it is co-expressed with spalt and the lncRNA ivory. pdm3 is also necessary for determining wing margin stripes, where it is again co-expressed with spalt, leading us to propose that the eyespot and wing margin gene regulatory networks could be homologous. Finally, pdm3 affects pigmentation of the ventral hindwing, phenocopying the seasonally-plastic color switch in J. coenia. Together, our work shows that POU domain transcription factors play diverse roles in butterfly wing pattern development and highlights nubbin as one of the first genes implicated in the repressive function of wing veins in color pattern determination. Highlights- Gene expression and knockouts reveal three POU factors regulate butterfly wing color pattern - nubbin regulates eyespot development, likely via a repressor from the wing veins - nubbin controls scale color and morphology across the wing - pdm3 coordinates eyespot development and is co-expressed with spalt and ivory - Expression of genes in the eyespot and wing margin suggests network homology

Cohesin is a large multisubunit protein complex that plays essential roles in cell proliferation, genome organisation, and gene regulation in metazoans. Germline mutations in cohesin subunits or regulators cause a group of human developmental disorders collectively known as cohesinopathies. Increasing evidence indicates that individual cohesin subunits can confer distinct molecular functions to the complex; for example, STAG1 and STAG2 have both overlapping and non-overlapping roles in genome organisation. The zebrafish tailbud provides an excellent developmental model for investigating the coordination of cell proliferation and differentiation, processes in which cohesin has crucial functions. We previously demonstrated that loss of Stag2 disrupts Wnt signalling and mesoderm patterning in the zebrafish tailbud. Here, we show that, unlike mammals, zebrafish can tolerate complete loss of Stag1 from embryogenesis through to adulthood. In contrast to Stag2 deficiency, loss of Stag1 impairs cell cycle progression, activates p53 signalling, and induces a metabolic shift towards catabolism. BMP signalling is reduced in Stag1-deficient embryos and is accompanied by expansion of BMP antagonist chordin expression. Stag1 loss also alters chromatin accessibility at the chordin locus and affects accessibility at chromatin domain boundaries. We propose that modulation of growth and signalling pathways compensates for the absence of Stag1, allowing embryonic development to proceed correctly. Together, these findings reveal distinct contributions of Stag1 and Stag2 to cell-cycle regulation, chromatin architecture, and developmental signalling during vertebrate embryogenesis.

Traumatic brain injury (TBI) impacts millions of individuals annually causing death, disability, and a heightened risk for long-term neurological and neuropsychiatric disorders. In recent years the fruit fly, Drosophila melanogaster has become a valuable model organism to study the cellular and molecular responses following TBI. AP-1 mediated transcriptional responses to TBI have previously been identified in Drosophila using pan-glial approaches. Fruit flies possess multiple glial subtypes which vary greatly in both cellular morphology and function, including glia of the blood hemolymph barrier, cortex, astrocyte-like, and ensheathing glia. By generating and utilizing a nuclear localized AP-1 transcriptional reporter, we identified glial subtype-specific differences in the extent of AP-1 activation following injury. Our findings identify a strong AP-1 response in the blood hemolymph barrier and ensheathing glia, a moderate response in cortex glia and little to no AP-1 activation in astrocyte-like glia. In addition, we inhibited AP-1 signaling in each glial subtype and tested the effect on acute survival. We found that inhibition of the AP-1 response in neuropil ensheathing glia leads to increased mortality following mild and moderate TBI. These results show that AP-1 activation levels vary across glial subtypes after TBI, with activation in neuropil ensheathing glia having a particularly important role in promoting post-injury survival. ARTICLE SUMMARYUsing Drosophila as a model organism, we investigated the early molecular and cellular response to traumatic brain injury. Our findings substantiate the requirement of a functional glial associated AP-1 transcriptional activation response for survival. Using colocalization studies, we characterized the AP-1 glial response in six morphologically and functionally distinct glia subtypes. After TBI, we find high levels of AP-1 activation in glia of the hemolymph brain barrier, cortex glia, and ensheathing glia. We further show the importance of AP-1 transcription within the neuropil ensheathing glia subtype for optimal survival following TBI.

Wounds represent compromised tissues that are susceptible to opportunistic infections. Tissue factors predisposing to wound infection include edema, poor vascularity, tissue hypoxia, necrosis, and wound size. However, it is unclear whether factors external to the tissue play a role. Detection of osmolarity imbalances between internal and external environment has been shown to alter epithelial wound closure and immune cell recruitment at injury sites. However, it remains unclear whether these osmotic surveillance pathways affect susceptibility to pathogenic wound infections. Here, we use live imaging and disease monitoring in zebrafish larvae to understand susceptibility of wound infection towards Pseudomonas aeruginosa. We show that exposure of wounds to isotonic solution results in enhanced susceptibility and pathogen burden. Furthermore, we demonstrate that this higher propensity for infection depends on osmolarity-mediated changes in wound sealing and neutrophil recruitment during the early stages of the wound response. These findings inform the design of experimental wound infection models as well as the clinical management of wounds.