Biology Open

The hallmark of sessile adult ascidians is a vase-like shape with a single oral and atrial siphon. Ciona, however, develops two atrial siphons after metamorphosis, which subsequently fuse into one. The mechanisms underlying this fusion are unknown. This study suggests that Hox2 controls this process. Hox2-knockout animals using Transcription-Activator-Like Effector Nuclease (TALEN) retain two atrial siphons throughout their lives. During normal fusion, epidermal cells between the siphons flatten along the anterior-posterior axis. This cellular flattening does not occur in Hox2-knockout animals, suggesting that the shape change in the epidermal cells produces tension, allowing the atrial siphon openings to converge at the midline for fusion. Hox2-knockout animals lack cupular organs, which are suspected hydrodynamic sensors in the internal epithelium of the fused atrial siphon and on the sperm duct. Among several knockout attempts, atrial siphon fusion was reproduced by only one TALEN pair, suggesting that this phenotype is driven by a mutation having a broader effect than those abolishing protein function. Many ascidians, unlike Ciona, develop a single atrial siphon shortly after metamorphosis. Our findings suggest that a phylogenetically conserved gene, Hox2, establishes this group-specific atrial siphon formation mechanism in Ciona.

High-resolution 3D imaging is an important strategy for visualizing and analysing complex skeletal tissue architecture and the bone marrow microenvironment. However, multicolor immunolabeling and imaging of intact skeletal tissues are technologically challenging. The current immunolabeling and clearing methods for intact skeletal elements are very limited, time-consuming and generate low-resolution data or depend on the use of reporter mice. Here, we describe a protocol for efficient clearing and immunolabeling of intact calcified tissues that enables superfast, single-cell resolution, and quantitative 3D light-sheet imaging of intact skeletal elements and teeth. A key aspect of our protocol is the addition of a collagenase digestion step after fixation and decalcification. This step enhances antibody penetration, resulting in deep, comprehensive staining throughout immunostained bones and other calcified tissues. The protocol includes soft tissue removal, fixation, decalcification, bone dehydration, and bleaching, followed by antigen retrieval and permeabilization before the collagenase digestion step. This procedure is performed to prepare the samples for the tissue clearing process that improves bone tissue transparency prior to light-sheet imaging. The entire protocol, from bone collection to image analysis and quantification, takes about 4 days to complete, thus offering significant improvements over previous methods. This protocol is broadly applicable to the visualization of bone microstructure, bone marrow analysis, vascular and neural network mapping, and the study of signaling molecules in bone development and growth. The protocol requires experience with standard tissue processing and immunostaining techniques, and prior experience in tissue clearing and light-sheet imaging is beneficial but not essential. Key pointsO_LIA protocol for efficient clearing and immunolabeling of intact calcified tissues that enables superfast, high-resolution, and quantitative 3D imaging of various intact bones and teeth. C_LIO_LIThe entire protocol takes only 4 days to complete the comprehensive staining and perfect transparency throughout the intact bones, offering significant improvements over previous methods. C_LI Key referencesBiswas, L. et al. Cell 186, 382-397.e24 (2023): https://doi.org/10.1016/j.cell.2022.12.031

BackgroundThe fission yeast stress-activated protein kinase (SAPK) pathway includes a conserved mitogen-activated protein (MAP) kinase cascade that regulates multiple cellular processes and is activated by several types of external stress. Understanding how Sty1, the MAP kinase in the SAPK pathway, controls these processes is complicated by the fact that different stressors can have stressor-specific effects that may be difficult to separate from the effects of Sty1 activation itself. Moreover, upon stress, Sty1 activation is usually short-lived. Previously, we developed a fission yeast strain, SISA, in which Sty1 kinase activity can be switched on in a sustained manner in the absence of external stress. This required combining multiple mutations in the SAPK pathway, including an analog-sensitive version of Sty1. When SISA cells are grown in the presence of analog-sensitive kinase inhibitors, Sty1 is inhibited, but when inhibitor is removed, Sty1 becomes hyperactive. While this strain was useful, it had several limitations. ResultsHere we describe and validate a more rationally-designed strain, SISA4, that retains the features of the original SISA strain while overcoming its limitations. SISA4 is more stable genetically than SISA, easier to use in genetic crosses, and easy to identify by phenotype or genotyping. We show that analog-sensitive kinase inhibitors 4-Amino-1-tert-butyl-3-(1-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1) and 4-Amino-1-tert-butyl-3-(3-bromobenzyl)pyrazolo[3,4-d]pyrimidine (3-BrB-PP1) are equally potent for inhibiting analog-sensitive Sty1 in vivo, and we determine optimal inhibitor concentrations for converting SISA4 cells from a Sty1-inhibited state to a Sty1-hyperactive state. We also find that both 1-NM-PP1 and 3-BrB-PP1 have measurable off-target effects in wild-type cells, although these are modest and generally do not affect interpretation of experiments. Finally, using SISA4, we show that the Sty1-activated transcription factor Atf1 plays an unexpected role in maintaining cell-polarity disruption after Sty1 hyperactivation. ConclusionsSISA4 will be useful for investigating how SAPK pathway activation regulates diverse cellular processes.

TULIP (TUbular LIPid binding) domain proteins (TDPs) are found in all living organisms including bacteria. They have various documented functions, some of which clearly related to their intra- or extracellular lipid transfer activities. Extracellular, BPI-related TDPs of insects (B-TDPs, also known as Takeout-related proteins), are often found in chemosensory organs, but little is known regarding their exact location or how they could contribute to olfaction or gustation. We have surveyed and updated the full set of Drosophila B-TDPs and found that roughly 50% are overexpressed in chemosensory organs. Focusing on three genes clustered on the third chromosome, we provide evidence that at least one of the encoded proteins is secreted in the lymph cavity housing the dendrites of olfactory neurons. Biochemical data give support for a putative function of B-TDPs as odorant transporters, but loss-of-function analyses also hint to a potential role as a barrier against plant-emitted terpenoids.

Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. Key PointsO_LIzebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages C_LIO_LIzebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis C_LI

Microproteins translated from short open reading frames are increasingly understood to play important roles in cell biology and development. Here, we describe a microprotein in Drosophila that is expressed in ovarian follicle cells which surround the developing oocyte. The Dafcin microprotein is predicted to form an amphipathic alpha-helix, a structure known to interact with lipid bilayers. The structure of Dafcin most resembles the influenza HA fusion peptide, which induces negative curvature of endosomal membranes. Dafcin tagged with GFP localizes to the Golgi and is ultimately secreted from the follicle cells. Remarkably, this occurs without the microprotein having a secretory signal sequence. The protein is taken up into the oocyte by endocytosis, localizing to the inner face of storage lysosomes called yolk granules. Mutant analysis shows that Dafcin is required to limit the size of yolk granules. This may occur by inducing negative membrane curvature like HA peptide. In support, liposomes formed in vitro with both Dafcin and HA peptides are smaller in size.

UNC-6/Netrin is a conserved regulator of dorsal-ventral axon and cell migrations. UNC-6 is composed of a Laminin N-terminal domain (LN), three epidermal growth factor repeats (EGF), and a Netrin C terminal domain (NC). Here, we identified missense mutations in distinct UNC-6 domains and assessed their roles in dorsal VD/DD motor axon guidance and ventral AVM axon guidance. A missense mutation in a conserved residue of the LN domain (G289D) resulted in dorsal and ventral axon guidance defects similar to unc-6 null. A distinct missense mutation in the LN domain (S120F) was hypomorphic and strongly perturbed ventral AVM axon guidance with minimal effects on dorsal VD/DD axon guidance, showing that S120F is predominantly required for ventral guidance. Missense mutations altering conserved cysteine residues involved in di-sulfide bonding in the EGF domains were analyzed. EGF1(C321G) caused both ventral and dorsal axon guidance defects albeit weaker than unc-6 null, indicating that EGF1 is required for both. EGF2(C347Y) strongly affected dorsal VD/DD axon guidance similar to unc-6 null, with weaker perturbation of ventral AVM axon guidance. Previous results revealed that EGF3(C410Y) specifically disrupted dorsal axon guidance, a result that we confirmed. Our studies using missense mutations in the endogenous unc-6 locus complement previous structure-function studies using transgenic expression, and identify domains specifically required for ventral AVM guidance (S120Y in the LN domain) and dorsal VD/DD axon guidance (C410Y in EGF3). The crystal structure of UNC-6 indicates conserved N-linked glycosylation at N114 and N128. Mutation of these sites in UNC-6 had no effect on dorsal ventral axon guidance, showing that they do not play a major role. However, the N114 and N128 mutations interacted genetically with unc-40 and unc-5 mutations, indicating that these glycosylation sites indeed have a role in UNC-6 signaling. Our results will inform studies on how these distinct UNC-6 domains interact with guidance receptors (e.g. UNC-40/DCC and UNC-5) and other extracellular molecules to mediate dorsal-ventral axon guidance.

Periodic patterns are a frequent motif in biology that occurs across diverse tissues and species. In mammals, pigmentation patterns such as zebra stripes or tiger stripes are well-known examples of periodic patterns; more recently, the myenteric plexus (MP) of the enteric nervous system (ENS), which controls gastrointestinal motility, has been found to exhibit a striped organization in humans and laboratory mice. In domestic cats and other felids, the Transmembrane aminopeptidase Q (Taqpep) gene plays a key role in color pattern establishment during skin development, but its patterning role has not been examined in other tissues. Here, we show that, in laboratory mice, Taqpep is required for normal patterning of developing hair follicles and the MP. Using both sequencing and histologic techniques, we found Taqpep is expressed in mesenchymal cells in embryonic skin and intestine directly adjacent to where periodic patterning occurs. We generated Taqpep mutant mice, which exhibit disrupted epidermal patterning akin to the changes in periodic coat patterning observed in Taqpep mutant cats. The intestine of Taqpep mutants has irregularly periodicity of enteric neuronal stripes, and enteric neurons in Taqpep mutants exhibit disrupted Wnt signaling. This work provides new insight into the mechanism of enteric neuronal patterning and identify Taqpep as a common and conserved mediator of periodic patterning across mammalian tissues and organisms. Author summaryPeriodic patterning is a frequent motif in biology. Examples include pigmentation patterning such as tiger stripes and, as recently identified in both mouse and human, the striped organization of enteric neurons in the myenteric plexus of the intestine. In domestic and wild cats, the Transmembrane aminopeptidase Q (Taqpep) gene is essential for the establishment of periodic patterning. Whether this gene plays a conserved role in periodic patterning across other tissues and species has yet to be explored. We found that Taqpep is expressed in mesenchymal cells in embryonic mouse skin and intestine at key locations and developmental stages to instruct periodic patterning. We next generated Taqpep mutant mice that exhibit disrupted periodic patterns in both developing skin follicles and in enteric neuron organization. Thus, Taqpep is essential in establishing periodic patterning in diverse mammals and tissues.

Survival analysis is a workhorse assay in Drosophila research to evaluate somatic fitness. It is indispensable in the study of ageing and insightful in immunity, metabolism, radiobiology, toxicology, ecology, and others. While conceptually simple, lifespan measurement is labour-intensive because it requires the continuous manual maintenance of large experimental cohorts. Here, we describe Drosben, an approach that combines a 3D-printed device to transfer flies from several vials simultaneously, a paper system for quick data recording and accompanying software that automatically digitalises life tables for analysis. We show that using Drosben reduces the time investment to perform lifespan assays by ~85%, with improved speed regardless of experience handling Drosophila vials. Using Drosben, we address the effects on longevity of chronic feeding of indole-acetic acid (IAA), naphthalene-acetic acid (NAA) and trimethoprim (TMP) -- compounds used to control heterologous targeted protein degradation systems. We find that IAA and NAA have noticeable deleterious effects while TMP has a small protective effect specifically in females. We further show that strong static magnetic fields do not affect Drosophila lifespan. Our work suggests that Drosben can cheaply accelerate research where lifespan is used as a life history trait.

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

Histone proteins, which reside in the nuclei of eukaryotic cells, are involved in diverse cellular processes. The core histone H4 serves as a structural component of the nucleosome. Patients carrying mutations in H4Clustered histone (H4C) genes exhibit a broad spectrum of developmental abnormalities, including short stature, microcephaly, intellectual disability, growth retardation, and digital anomalies. However, the impact of H4 mutations on mammalian embryogenesis remains largely unclear. Here, we demonstrate that histone H4C genes play crucial roles in skeletal development and cortical neurogenesis. We found that mRNAs of the histone H4C gene family are specifically expressed in proliferating progenitor cells in the developing mouse neocortex and in human induced pluripotent stem cell-derived cortical organoids. CRISPR-mediated disruption of H4C3 in mice caused severe defects in skeletal formation and neocortical neurogenesis. Furthermore, overexpression of a mutant form of H4C3 resulted in altered expression of genes associated with cellular migration and motility. Together, these findings suggest that histone H4 plays a critical role in regulating the balance between proliferation and differentiation during mammalian embryonic development, thereby explaining the broad spectrum of patient phenotypes.

Photoreceptor outer segments are sensory cilia whose maintenance depends on a balance between basal disc renewal and tip shedding, controlled by intraflagellar transport and axonemal microtubule organization. Microtubule plus-end proteins regulate microtubule dynamics and are strong candidates for roles in this process. In this study, mCherry-tagged EB1, EB3, and DCX were overexpressed in zebrafish (Danio rerio) cone photoreceptors under a cone-specific promoter. Eyes were examined at 5 and 10 dpf, and eyecup depth, diameter, and cone photoreceptor area were quantified relative to uninjected controls. At 5 dpf, all three constructs produced eyes indistinguishable from those of controls. By 10 dpf, all three constructs significantly increased eye cup depth and cone photoreceptor area. EB1 and DCX also significantly increased eye cup diameter. EB1 and, more severely, EB3 also caused retinal holes, mainly in the retinal pigment epithelium and at the outer nuclear/outer plexiform layer, along with misshapen cells near the inner plexiform layer. DXC did not cause retinal holes, but, like EB1 and EB3, produced enlarged, bulbous cone outer segments. The results show that overexpression of any of the three +TIPs results in a similar eye and photoreceptor overgrowth phenotype, while also producing construct-specific defects: EB1 and EB3 disrupt the broader retinal architecture, whereas DCX produces enlarged eyes. The shared outer segment hypertrophy suggests an imbalance between cargo delivery at the basal end and shedding of the distal tips. The organomegaly may reflect altered progenitor signaling in the ciliary marginal zone.

Effective communication between cells is essential for the typical development and behaviour of an organism. In this context, gap junctions represent the most universally preserved components at cellular membranes of multicellular organisms, facilitating metabolic and electrical connections between cells. Disruptions in these junctions have been linked to various developmental abnormalities and pathological conditions in humans. The invertebrate gap junction proteins, referred to as innexins, exhibit conserved cellular and molecular mechanisms of functioning with their vertebrate counterparts, known as connexins. Consequently, they provide valuable means for studying and understanding the functions of gap junctions in development. In the Drosophila embryo, innexin-2 is expressed in the amnioserosa and ectoderm, where it is required for epithelial morphogenesis. Genetic depletion of innexin-2 results in cuticular defects and embryonic lethality. Pannier, a GATA family transcription factor, is a key regulator of dorsal tissue development in Drosophila and is expressed in the amnioserosa, dorsal ectoderm and the dorsal vessel during embryogenesis. Pannier mutants exhibit defects in dorsal closure, cuticle formation, and cardiac specification. Although substantial evidence from vertebrate systems indicate that connexin expression is regulated by transcription factors such as GATA4, Nkx2.5, Tbx2, Tbx3, and Tbx5, whether a similar regulatory relationship exists between these transcription factors and gap junction proteins in Drosophila remains unknown. In this study, we investigate how innexin mediated intercellular communication impacts pannier dependent morphogenetic processes during Drosophila embryogenesis.

Many insects enter diapause, a programmed state of developmental arrest that enables survival under adverse environmental conditions. In Drosophila melanogaster Meigen, 1830, diapause is characterized by reproductive arrest and reduced intestinal growth, accompanied by suppressed intestinal stem cell (ISC) activity. Juvenile Hormone (JH) promotes ISC proliferation under favorable conditions, but its capacity to modulate stem cell dynamics during cold-induced diapause remains unclear. Here, we investigated whether JH signaling can reactivate midgut remodeling in adult females maintained at 11. At this temperature, flies exhibited pronounced gut atrophy and elevated Phospho-histone H3 (PH3+) cell abundance, consistent with temperature-dependent G2/M phase arrest JH treatment significantly increased the proportion of Delta-positive progenitor cells in the anterior (R2) and posterior (R5) midgut regions at both 11 and 25, demonstrating that JH acts as a conserved mitogen for the ISC pool irrespective of thermal environment. A trend toward reduced PH3+ accumulation in the posterior midgut following JH treatment (p = 0.061) suggests possible facilitation of mitotic exit, though this effect did not reach statistical significance. Despite cellular-level changes, JH treatment did not restore overall gut size, indicating that the 72-84 hour exposure window was insufficient for subsequent tissue hypertrophy. Additionally, we identified a recurrent cold-induced pathology of gut distension, provisionally termed Lumen Obstruction Syndrome (LOS), which was independent of JH signaling. These findings reveal an uncoupling of JH-driven stem cell expansion from gross organ growth under diapause conditions, highlighting the selective sensitivity of the ISC compartment to endocrine signaling during environmental stress.

Reproductive isolation is a central driver of speciation and can be reinforced by both innate mate preferences and mate preference learning. Pigmentation genes are strong candidates for pleiotropic effects on these processes because they shape visual traits used in mate choice and may also influence neural function, yet their role in learning is poorly understood. Here, we test whether the pigmentation gene yellow affects innate mate preference and aversive mate preference learning in female Bicyclus anynana, and whether these effects are associated with changes in brain dopamine levels. Using CRISPR-Cas9, we knocked out the yellow gene, developed a mutant line, and tested the mate preferences and mate preference learning ability of mutant females compared to wild-type (WT) females. We find that loss of yellow does not alter assortative mating based on pigmentation or disrupt innate visual or olfactory preferences. In contrast, loss of yellow does influence learning ability, as mutant females failed to modify mate preference following aversive premating experience, indicating an impairment in aversive learning. Despite yellows role in the melanin biosynthesis pathway, brain dopamine levels remain unchanged in mutant females relative to WT females. These findings identify yellow as a pleiotropic gene influencing both pigmentation and aversive mate preference learning, providing evidence that pigmentation genes can shape behavioral processes important for reproductive isolation and speciation. Significance statementPigmentation genes are best known for controlling color patterns, but they may also influence behavior through shared neural pathways. Here, we show that the pigmentation gene yellow is required for aversive mate preference learning in the butterfly Bicyclus anynana. Females lacking yellow retain normal innate visual and olfactory mate preferences but fail to learn to avoid previously unattractive mates. Surprisingly, this learning deficit is not associated with altered dopamine levels, suggesting that downstream neural signaling pathways are involved instead. These findings identify a gene that influences both pigmentation and learned mate preference, providing evidence that pigmentation genes can shape behavioral processes important for reproductive isolation and speciation.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

The preparation of multi panel figures remains a labor intensive step in scientific publication. Albeit there are specific tools available to solve this problem, they are often highly specialized, difficult to install, or time consuming to learn. Griphus is a standalone graphical application designed for rapid composition and experimentation with multi panel figures, developed by and for zoological taxonomists. Functions specifically designed for multi panel composition include automatic figure numbering and placement, aspect ratio operations, spacers, layout rotation, layout suggestions, and automatic generation of figure legends, including scale bar descriptions. The software can perform both spatial interpretation of images on the canvas and work with a simple, editable layout formula. It also enables instant multi panel composition, with numbered images and automatic contrast selection for the numbers, obtained simply by loading images. User defined parameters such as target printable dimensions, resolution, spacing, and color mode are preserved throughout the work. The program produces coordinated outputs consisting of the final composite figure, a readable file describing the layout structure, and a .gri file storing images, transformations, and parameters for exact regeneration. Griphus is intended as a complementary tool to professional image software, providing a simple and efficient environment for constructing high quality multi panel figures.

The endoderm is specified at the onset of gastrulation and subsequently undergoes extensive migration before forming an epithelial sheet that gives rise to multiple organs, including the gut and respiratory tracts. Although the gene regulatory network underlying endoderm specification and the later processes that regionalize the gut are increasingly well understood, comparatively little is known about the intervening developmental events. Using single cell transcriptomics, we profiled the zebrafish sox17 lineage, comprising endoderm and dorsal forerunner cells, throughout and immediately after gastrulation. We found that dorsal forerunner cells remain transcriptionally homogeneous while undergoing coordinated temporal changes, associated with ciliogenesis and epithelial organization, during assembly of Kupffers vesicle. In contrast, endoderm cells transition from a migratory to an epithelial transcriptional state while progressively acquiring distinct regional identities. These findings indicate that endoderm regionalization emerges within the context of a broadly shared transcriptional program associated with migration and epithelialization.

The electrophysiological process of auditory transduction in insects remains largely conjecture due to the unknown role of ion channels localised to the cilia, but experimental evidence supports either NompC or Nan-Iav as the auditory mechanotransduction ion channel. Here, we knocked down two key genes that code for the two candidate sound-activated ion channels using dsRNA-mediated RNA interference. We measured sound-evoked activity of the auditory nerve and intracellular electrical currents from the ciliated ending of individual auditory receptors. We found that the sound-evoked nerve activity was reduced in nompC, nan and ift88 knockdown. Using whole-cell patch clamp recordings we found that nompC and nan knockdown resulted in reduced sound-evoked transduction current. Stochastic depolarisations hypothesised to be mediated from one of the candidate mechanotransduction ion channels, either NompC or Nan-Iav, where not affected by knockdown of either channel. The discrete depolarisations are therefore mediated through another unidentified ion channel. We test the hypothesis that discrete depolarisations are graded action potentials that travel toward the soma through noise analysis of the transduction current and analysis of discrete depolarisations to voltage-steps. As a positive control we also knocked down ift88, a protein essential for transporting proteins, including ion channels, along the cilium and found both the transduction current and the discrete depolarisations decreased. Key pointsO_LIInjection of dsRNA decreased RNA of nompC and nan C_LIO_LISound-evoked nerve activity is reduced for RNAi-mediated knockdown of nompC and nan C_LIO_LINompC and Nan both contribute to the transduction current C_LIO_LIThe stochastic discrete depolarisations are not due to NompC or Nan-Iav ion channel but to a third unidentified ion channel. C_LIO_LINoise analysis of the transduction current and the discrete depolarisations suggests they are graded action potentials that travel in the direction of the soma. C_LIO_LIKnockdown of ift88 reduced both the transduction current and discrete depolarisations. C_LI Significance StatementInsects are important to understand, economically, agriculturally and medically. However, we still do not understand fundamental aspects of how insects detect their own body movements, vibrations and sound. These senses are detected by insect chordotonal organs, specialised miniaturised mechanoreceptors that convert movements into electrical signals through specialised ion channels. Previous experimental work has advocated either NompC or Nan-Iav as the mechanosensitive ion channel. Here, for the first time, we reduced the expression of both nompC and nan and measured the sound-evoked transduction current directly from neurons in a specialised auditory chordotonal organ. In contradiction to previous studies, we show that both ion channels contribute to the transduction current and find that electrical signals termed "discrete depolarisations" travel toward the soma.

Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.