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.

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.

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.

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.

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.

Genetic lineage tracing is a powerful approach for investigating cell fate and tissue development, but versatile Cre/loxP-based tools remain limited in avian models. Here, we generated and characterised a Cre-responsive transgenic quail line that enables permanent genetic labelling of embryonic cell populations. The reporter line ubiquitously expresses nuclear-localised mCherry, which is excised following Cre-mediated recombination, enabling expression of nuclear-localised EGFP. We demonstrate that this reporter can be coupled to multiple Cre delivery strategies, providing flexible spatial and temporal control of lineage labelling. Ubiquitous or cell-type-specific Cre expression induces recombination and stable EGFP labelling that is maintained during clonal expansion. Optogenetic activation of a split Cre system enables highly spatially and temporally restricted recombination. Furthermore, lipid nanoparticle-mediated delivery of Cre mRNA enables efficient recombination in distinct embryonic cell populations, including mesodermal, neuroepithelial, neural crest, endothelial and circulating cells. This transgenic reporter quail provides a flexible platform for permanent lineage labelling without the need to generate tissue-specific Cre transgenic lines and can be readily combined with diverse Cre delivery strategies to investigate cell fate, lineage and morphogenesis in vivo.

1. For many animals, vision represents the primary sensory modality governing a vast diversity of behaviours. Creating 'virtual twins', or visually realistic digital reconstructions of real individuals interacting within their environments, offers a promising approach for understanding how visual information guides an animal's decisions, behaviours, and interactions. Such systems enable the creation of video scenes from any view, including the viewpoint of the animals themselves. This allows receiver information to then be analysed from the perspective of species-specific visual systems, or for the accurate reconstruction of a real animal's movement to provide social stimuli for behavioural experiments. 2. Despite advances in 3D modelling, photorealistic scene rendering, and animal tracking, efficiently creating realistic digital reconstructions of movement remains challenging for many species. In fishes, a continuously-deforming body due to the absence of discrete joints make it particularly difficult to map tracked body keypoints onto articulated digital models, often resulting in unnatural or erratic animations. 3. Here, we introduce DigiFish, an add-on for the open-source animating software Blender, that generates realistic fish animations from tracking data. By integrating anatomical keypoint tracking with spline-based body curvatures, DigiFish reconstructs biologically-realistic swimming postures and movements of fishes within customisable virtual aquatic environments. The system also supports virtual 'point-of-view' cameras that can be placed arbitrarily within a scene, or dynamically driven by eye-tracking data, enabling unlimited perspectives on how individuals, conspecifics, or landmarks appear against complex backgrounds and how these views change with realistic swimming and eye movements. 4. We provide the Blender add-on and user guide and use DigiFish to reconstruct two different contexts: staged predator-prey interactions in the laboratory and natural schooling of wild fish in the field. Using a further reconstruction of fish swimming in an arena, we also use an Elementary Motion Detector model to show that our virtual reconstructions successfully capture perceptually-realistic movements and postures of individual fish within a digitally reconstructed scene. 5. By integrating photo-realistic scenes with biologically-realistic, simulated motion, DigiFish offers a flexible and accessible platform for investigating visually guided behaviour, perception, and cognition in complex and dynamic environments.

JAK-STAT signalling pathway activation relies mostly on the transcriptional regulation of its ligands. The pathway, conserved in vertebrates and invertebrates, is required for the development and homeostasis of various organs. In contrast to the complexity of the vertebrate pathway, with a multiplicity of ligands, receptors, kinases and transcription factors, the simplicity of the Drosophila pathway with only three ligands (Upd1, Upd2 and Upd3), a single receptor (Dome), a JAK kinase (Hop) and a STAT transcription factor (STAT92E) facilitates the genetic study of its multiple functions. To discover how, when and where the Upd1 ligand performs each organogenetic function, we have searched for the different enhancers controlling its dynamic spatio-temporal regulation and used them to generate minigenes replicating specific stages of Upd1 expression. The combination of these minigenes with mutations deleting all three Upd ligands, allowed us to identify the timing when the various embryonic functions are mediated, including head involution, tracheae, corpora allata and prothoracic gland specification, posterior spiracle formation and hindgut elongation. We uncover most of the enhancers controlling STAT activation during embryonic organogenesis and those driving activation in the adult testis and the ovary. As expected, we identified redundant shadow enhancers but, interestingly, we found a different type of redundant enhancers that mediate identical developmental functions although driving completely different expression patterns. We name this new class of functionally redundant enhancers driving different expression as shade enhancers. We propose shade enhancers provide evolutionary plasticity as they could allow the pattern of gene expression to diversify without altering the underlying body plan.

Prospero-related homeobox (Prox) genes encode a family of transcription factors that play essential roles in the development of several organs and systems, including the central nervous system, lymphatic endothelium, musculature, and liver. Despite their developmental importance, the evolutionary history of the vertebrate Prox gene family remains poorly understood. In this study we combined phylogenetic and synteny analysis to characterise the evolution of the Prox family in vertebrates. Our results reveal that two to three Prox subfamilies were already present in the last common ancestor of jawed vertebrates. We clarify the identity and evolutionary relationships of well-studied members of this family and identify multiple independent losses of Prox2 and Prox3 genes in specific vertebrate lineages. Furthermore, we uncover evidence for the existence of a fourth Prox gene in the ancestral vertebrate genome, which was subsequently lost. Overall, this study provides the first comprehensive analysis of the evolutionary history of the vertebrate Prox gene family and establishes a foundations for future studies on the functional roles of these genes.

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

Background: Insufficient oxygen (hypoxia) or complete lack of oxygen (anoxia) causes cellular dysfunction and death in terrestrial animals. As a result, animals have developed homeostatic strategies, including changes in movement strategies to escape and survive stressful environments. Caenorhabditis elegans respond to changes in oxygen by increasing the frequency of reversals (backward movement) and increasing locomotor speed; these behaviors enable them to escape unfavorable environments and move to more suitable ones. The molecular and neural mechanisms underlying behavioral responses to low-oxygen environments are incomplete. Neuropeptides regulate C. elegans behaviors by modifying neural circuit function in response to hypoxia. Loss of function in C. elegans neuropeptide nlp-40 and its receptor aex-2 increased survival to anoxia [1]. However, the cells in which NLP-40 and AEX-2 function for anoxia, as well as the mechanisms underlying responses to anoxia, remain elusive. We hypothesized that NLP-40 and AEX-2 regulate anoxia by altering escape behaviors. Results: We found that neuropeptide NLP-40 functions in cholinergic neurons and AEX-2 functions in cholinergic or GLR-1 interneurons or motor neurons to regulate susceptibility to anoxia. We found that loss of either nlp-40 or aex-2 decreased locomotor activity and reversal frequency early during 48 hours of anoxia, suggesting that nlp-40 and aex-2 are required for the proper execution of these behaviors. We found that neuronal knockdown of nlp-40 recapitulated the behaviors observed in nlp-40 mutant animals and increased survival; however, restoring aex-2 function in glr-1 interneurons or motor neurons was sufficient to rescue behaviors to wild-type and restore vulnerability to anoxia. Conclusions: Overall, our results suggest that NLP-40 and AEX-2 likely function in cholinergic or glr-1 interneurons or motor neurons to regulate escape behaviors during anoxia. Escape behaviors are part of the homeostatic response and are typically protective in the wild; however, in our paradigm, we show that loss of either nlp-40 or aex-2 reduces escape responses and confers protection against 48 hours of anoxia. Collectively, these results highlight a new role for NLP-40 and AEX-2 in anoxia and shed light on mechanisms underlying the adaptive responses to anoxia.

Stem cell factor (SCF) from Leptin Receptor-expressing (LepR+) mesenchymal stromal cells and endothelial cells is required for hematopoietic stem and progenitor cell maintenance in bone marrow. Consistent with this, Scf is highly expressed by LepR+ cells and at lower levels by endothelial cells in bone marrow. However, endothelial cells outside the bone marrow produce circulating SCF, raising the question of whether the SCF that promotes stem and progenitor cell maintenance is produced inside or outside bone marrow. We found that Scf deletion in spleen and liver endothelial cells reduces SCF levels in the blood without affecting stem or progenitor numbers in the bone marrow. Moreover, deletion of Scf from mesenchymal cells in the long bones, but not vertebrae, of Prx1Cre; Scf fl/fl mice depletes stem and progenitor cells in long bones but not vertebrae. SCF thus acts locally within the bones in which it is made to promote stem and progenitor cell maintenance.

Intestinal epithelial barrier is a key feature of the healthy gut, and disruption of this barrier is a driver of mucosal inflammation. Integrity of the intestinal epithelial barrier is controlled by tight junctions (TJs) and adherens junctions (AJs) associated with the underlying actin cytoskeleton. Posttranslational modifications of actins affect the cytoskeletal assembly and dynamics, however whether such modifications regulate epithelial barriers remains unknown. SETD3 methyltransferase methylates actins at the His73 residue, thereby enhancing filamentous (F)-actin polymerization and turnover. This study examines the role of SETD3 in regulating structure and function of normal intestinal epithelial barrier, as well as its involvement in barrier disruption during mucosal inflammation. siRNA mediated knockdown of SETD3 in DLD-1 and HCA-7 human intestinal epithelial cells (IEC) resulted in several functional and morphological cell abnormalities. These include increased permeability of the IEC barrier, diminished assembly of the perijunctional F-actin belt, increased cell size, and delayed TJ reassembly during calcium switch. Consistently, SETD3 knockout (KO) mice displayed increased intestinal permeability and decreased labeling intensity of apical {beta}-actin as compared to wildtype controls. Furthermore, SETD3 KO mice showed increased cell stemness and proliferation in colonic epithelium without defects in AJ/TJ assembly and secretory cell differentiation. In addition, SETD3 KO mice developed more severe disease symptoms during dextran sodium sulfate (DSS)-induced colitis, which were accompanied by accelerated mucosal erosion and increased macrophage, T cell and neutrophil infiltration into colonic tissue. Overall, our data suggests that SETD3 acts as a novel positive regulator of the intestinal epithelial barrier and plays a protective role during intestinal mucosal damage and inflammation.

Coleoid cephalopods (cuttlefish, octopus, and squid) are marine mollusks with elaborate nervous systems that support a diverse repertoire of complex behaviors. These include the neural control of the color, pattern, and texture of the skin, facilitating both adaptive camouflage and innate patterning that may reflect internal state. The development of transgenic cephalopods expressing fluorescent proteins, optogenetic actuators, and reporters of neural activity would contribute a new and important technology to cephalopod biology. The generation of transgenic cephalopods, however, has remained a major challenge. Here, we report the development of stable transgenic dwarf cuttlefish (Ascarosepion bandense) expressing ubiquitous nuclear-localized mScarlet, a red fluorescent protein. We evaluated multiple strategies for transgenesis, and established cuttlefish lines using both CRISPR and the transposons Sleeping Beauty and Minos. The stable expression of transgenes enabled live imaging of cell dynamics during embryonic development. The Minos transposon emerged as the most efficient transgenesis strategy and is adaptable to promoters and transgenes of choice. These strategies now enable the generation of diverse genetic tools for mechanistic studies of cephalopod biology.

Ultraviolet-C (UV-C) radiation is a potent environmental stressor that induces DNA damage, accumulation of reactive oxygen species (ROS), and cellular dysfunction. This also initiates coordinated molecular responses to regulate cellular adaptation and recovery. Small extracellular vesicles (sEVs) have emerged as key mediators of intercellular communication through the selective incorporation of microRNAs (miRNAs), proteins, and lipids. However, the temporal changes of sEV-associated cargo during the progression from UV-C-induced damage to recovery remain uncharacterized. We hypothesised that UV-C exposure induces stage-specific changes in sEV cargo (miRNA and proteins) that promote the transition of cells from damage to the response and recovery phases. To examine this, a time-resolved UV-C exposure model was established using Caenorhabditis elegans (C. elegans). In this paper, we studied 2.5 hours of UV-C exposure, including cellular damage, intracellular ROS levels, and DNA integrity, to define the damage and repair state. Based on these temporal variations, samples were collected: i) immediately after exposure, ii) 6 hours post-exposure, and iii) 16 hours post-exposure were designated as damage, response, and recovery conditions, respectively. sEVs isolated from whole-worm preparations were characterised using biophysical and molecular approaches, followed by miRNA sequencing and proteomic analyses. Integrative analysis further identified miRNA-protein associations and associated them with pathways involved in stress adaptation, metabolism, signal transduction, and cellular homeostasis. Collectively, these findings provide a temporal molecular framework of sEV cargo associated with UV-C-induced damage, response, and recovery in C. elegans. Overall, this study identified candidate molecular signatures and miRNA-protein associations that underlie the temporally selective packaging of sEV-associated cargo following UV-C-induced damage, providing a basis for future tissue-specific and mechanistic investigations.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.

Hematopoietic stem cells (HSCs) arise from the hemogenic endothelium in the dorsal aorta through the endothelial-to-hematopoietic transition, a tightly regulated developmental process requiring coordinated signaling from the developing hematopoietic niche. Wnt and Notch signaling are essential for HSC specification, with Wnt functioning upstream of Notch; however, the molecular mechanisms by which these pathways are coordinated during HSC development remain poorly understood. Here, we identify Disheveled 2 (Dvl2), a key intracellular Wnt scaffold protein, as an essential regulator of HSC identity specification in zebrafish. Loss of dvl2 impaired HSC specification and reduced Notch signaling in the dorsal aorta, despite normal canonical Wnt activity in the developing endothelium, suggesting that Dvl2 regulates HSC specification independently of endothelial beta-catenin signaling. Hemogenic endothelial-specific expression of the Notch intracellular domain rescued HSC defects in dvl2 morphants, demonstrating that Dvl2 functions upstream of Notch activation in the hemogenic endothelium during HSC specification. In addition, loss of dvl2 reduced primitive macrophages and neutrophil populations. Restoration of primitive myeloid cells through gata1a knockdown rescued both endothelial Notch signaling and HSC defects, demonstrating that Dvl2 is also required for the supportive immune cells that induce HSC identity. Together, these findings reveal that Dvl2 coordinates hemogenic endothelial and immune niche cues to control HSC identity specification.

Reliable morphological criteria for sexing birds without the need for invasive procedures are scientifically and ethically valuable. Smith et al (2005) reported that iris colour assigns the genetic sex of Starlings Sturnus vulgaris with 98% accuracy, and bill base colour with 100% accuracy when birds are in breeding condition, but these figures have not been tested independently. We applied their criteria to 31 captive Starlings in breeding condition, using three observers blind to genetic sex, two of them new to Starling sexing. Bill base colour correctly predicted genetic sex for all 29 birds scored, with complete observer agreement. Mean iris score was correct for 27 of 31 (87.1%), fewer than the 94 of 96 reported by Smith et al (2005) for birds in breeding condition (Fisher exact test, P = 0.031), although with so few errors in either study this comparison is imprecise. Observer agreement was high, so these errors reflect iris colouration conflicting with genetic sex rather than inconsistent scoring; bill base colour was correct for all four misassigned birds. Body mass and tarsus length classified 77% and 58% of birds correctly, their published thresholds missing in opposite directions. We conclude that bill base colour should be the primary criterion for sexing Starlings in breeding condition; the accuracy of iris colour should not be assumed to be 98%.

Cortical positioning of oocyte meiotic spindles is required for reducing chromosome number by segregating chromosomes into polar bodies. In C. elegans, kinesin-1 transports membrane-bound organelles inward into a central ball that is hypothesized to push the meiotic spindle outward. Using focused ion beam scanning electron microscopy, we found that the percent volume occupied by membrane-bound organelles was 28% in a mature oocyte that has completed endocytosis of the yolk precursor, vitellogenin, compared to 17% in a less mature oocyte that has not yet endocytosed vitellogenin. We therefore hypothesized that blocking endocytosis of vitellogenin would reduce the size of the central ball of organelles packed inward by kinesin-1 and limit outward movement of the spindle. Depletion of vitellogenin or proteins required for endocytosis resulted in a smaller central volume of kinesin-coated organelles and defective spindle positioning. A computer simulation of a simple exclusion model faithfully recapitulated wildtype and mutant spindle position phenotypes. These results support a volume exclusion model for positioning acentriolar meiotic spindles.