Biology Open

The replenishment of specialized cells depends on the activity of stem cells. Recent advances in stem cell research have shown that the germline stem cells (GSCs) in Drosophila melanogaster can increase their mitotic activity in response to mating. Here, we show that this ability to respond to mating is eliminated if the males are mutant for the ABC transporter, White, the genetic background for a plethora of fly lines. Furthermore, we were not able to reproduce previous findings that female flies increase their GSC numbers and mitotic activity upon mating. Our findings underline the importance of careful experimental design and control specimen.

The transcription factor Odd-skipped has been implicated in many developmental processes in Drosophila melanogaster. Odd-skipped is expressed in a small cluster of neurons (Slater, Levy et al.) in the developing and adult CNS but its role in neurogenesis has so far not been addressed. Here we show that Odd-skipped plays a pivotal role in neurite growth and arborization during development. Loss-of-Odd-skipped function prevents neurite outgrowth whereas over and miss-expression causes neurite growth and arborization defects. In addition, miss-expression of Odd-skipped can induce cell death in some neural sub types. The neurite growth and arborization defects associated with Odd-skipped over expression correlates with a reduction in the pre-synaptically targeted protein Bruchpilot in axonal arbours suggesting an overall decrease in Odd neural synapse formation. This is supported by behavioural data showing that larvae in which Odd-skipped is overexpressed behave similarly to larvae in which Odd neurons are silenced showing that increasing Odd-skipped protein levels affect neural function. Finally, we demonstrate that using RNAi against Odd-skipped does not knock down Odd-skipped protein but instead cause an increase in protein levels compared to control larvae. This data demonstrates that RNAi can cause up-regulation of protein levels highlighting the importance of verifying protein levels when using RNAi approaches for knock-down.

IntroductionThe bicoid (bcd) gene in Drosophila has served as a paradigm for a morphogen in textbooks for decades. Discovered in 1986 as a mutation affecting anterior development in the embryo, its expression pattern as a protein gradient later confirmed the prediction from transplantation experiments. These experiments suggested that the protein fulfills the criteria of a true morphogen, with the existence of a homeodomain crucial for activation of genes along the anterior-posterior axis, based on the concentration of the morphogen. The bcd gene undergoes alternative splicing, resulting in, among other isoforms, a small and often neglected isoform with low abundance, which lacks the homeodomain, termed small bicoid (smbcd). Most importantly, all known classical strong bcd alleles used in the past to determine bcd function apparently do not affect the function of this isoform. ResultsTo overcome the uncertainty regarding which isoform regulates what, I removed the bcd locus entirely using CRISPR technology. bcdCRISPR eggs exhibited a short and round appearance. The phenotype could be ascribed to smbcd because all bcd alleles affecting the function of the major transcript, termed large bicoid (lgbcd) showed normally sized eggs. Several patterning genes for the embryo showed expression in the oocyte, and their expression patterns were altered in bcdCRISPR oocytes. In bcdCRISPR embryos, all downstream segmentation genes showed altered expression patterns, consistent with the expression patterns in "classical" alleles; however, due to the altered egg geometry resulting in fewer blastoderm nuclei, additional constraints came into play, further affecting their expression patterns. ConclusionsThis study unveils a novel and fundamental role of bcd in shaping the eggs geometry. This discovery demands a comprehensive revision of our understanding of this important patterning gene and prompts a reevaluation of past experiments conducted under the assumption that bcd mutants were bcdnull-mutants.

BackgroundA hallmark of arthropods is their segmented body, and the so-called Drosophila segmentation gene cascade that controls this process serves as one of the best-studied gene regulatory networks. An important group of segmentation genes is represented by the pair-rule genes (PRGs). One of these genes was thought to be the type-II transmembrane protein encoding gene Tenascin-m (Ten-m (aka odd Oz)). Ten-m, however, does not have a pair-rule function in Drosophila, despite its characteristic PRG-like expression pattern. A recent study in the beetle Tribolium castaneum showed that its Ten-m gene is not expressed like a segmentation gene, and hence is very unlikely to have a function in segmentation.\n\nResultsIn this study, I present data from a range of arthropods covering the arthropod tree of life, and an onychophoran, representing a closely related group of segmented animals. At least one ortholog of Ten-m/odz in each of these species is expressed in the form of transverse segmental stripes in the ectoderm of forming and newly formed segments - a characteristic of genes involved in segmentation.\n\nConclusionsThe new expression data support the idea that Ten-m orthologs after all may be involved in panarthropod segmentation.

Notch signalling is a critical regulator of multiple developmental processes through its ability to control gene expression and thereby influence cell fate specification and cell proliferation through direct cell-cell communication. Although Notch signalling has been implicated in myogenesis during late embryogenesis, its role in early mesoderm development has been largely unexplored. Endocytosis of the Notch ligand Delta and the Notch receptor extracellular domain, a critical step in Notch pathway activation, has been extensively observed in the ventral mesoderm of the early Drosophila embryo, indicating a potential for Notch signalling activity in this early germ layer. Here we present evidence that genes critical to mesoderm development require and are responsive to Notch signalling activity. Using a novel light-inducible Optogenetic variant of the Notch intracellular domain (OptoNotch), which affords precise spatial and temporal control over ectopic activation of Notch signalling, in combination with high-resolution fluorescent RNA in situ hybridization and qPCR, we identified a set of mesodermal genes whose expression is directly regulated by Notch signalling. We also provide evidence that Notch signalling indirectly regulates the dorsal-ventral patterning program mediated by the Toll signalling pathway through the Dorsal/ Twist/ Snail gene network. Our findings demonstrate that Notch signalling regulates ventral mesoderm patterning and is critical for establishing the mesoderm-mesectoderm-ectoderm boundary by regulating gene expression patterns and providing negative feedback on the upstream patterning network.

Background and objectivesPrevious work has identified that gene expression differences in cell adhesion pathways exist between humans and chimpanzees. Here, we used a comparative cell biology approach to assay interspecies differences in cell adhesion phenotypes in order to better understand the basic biological differences between species epithelial cells that may underly the organism-level differences we see in wound healing and cancer. MethodologyWe used skin fibroblast cell lines from humans and chimpanzees to assay cell adhesion and migration. We then utilized published RNA-Seq data from the same cell lines exposed to a cancer / wound-healing mimic to determine what gene expression changes may be corresponding to altered cellular adhesion dynamics between species. ResultsThe functional adhesion and migration assays revealed that chimpanzee fibroblasts adhered sooner and remained adherent for significantly longer and move into a "wound" at faster rate than human fibroblasts. The gene expression data suggest that the enhanced adhesive properties of chimpanzee fibroblasts may be due to chimpanzee fibroblasts exhibiting significantly higher expression of cell and focal adhesion molecule genes than human cells, both during a wound healing assay and at rest. Conclusions and implicationsChimpanzee fibroblasts exhibit stronger adhesion and greater cell migration than human fibroblasts. This may be due to divergent gene expression of focal adhesion and cell adhesion molecules, such as integrins, laminins, and cadherins, as well as ECM proteins like collagens. This is one of few studies demonstrating that these divergences in gene expression between closely related species can manifest in fundamental differences in cell biology. Our results provide better insight into species-specific cell biology phenotypes and how they may influence more complex traits, such as cancer metastasis and wound healing.

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C. elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells, and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C. elegans intestine, bublin recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1. In mouse intestinal organoids, bublin localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals. SummaryWe identify BBLN-1 as an evolutionary conserved regulator of lumen morphology in the C. elegans intestine. Loss of bbln-1 causes intermediate filament network reorganization that induces severe apical morphology defects. We also identify bublin (BBLN) as the mammalian ortholog, which can compensate for the loss of BBLN-1 in C. elegans.

Enteroblasts (EBs) are the cells responsible for the maintenance of the epithelium that lines the adult midgut in Drosophila. In response to cell death and damage, EBs undergo a Mesenchymal-Epithelial-Transition (MET) as they incorporate into the epithelium and differentiate into enterocytes (ECs). The morphogenetic mechanisms driving this MET process are not well understood. To improve phenotypic analysis of EBs, we established an analysis pipeline that uses machine learning segmentation to produce a reliable and automated quantification of cellular morphology and spatial distributions. EB morphology and fate is visualised using the Repressible Dual Differential stability cell Marker (ReDDM) approach. We show that wildtype EB cells exhibit a bimodal distribution pattern in which midguts fall into two categories: "quiescent" guts, in which EBs are evenly spaced out and newly formed ECs are uncommon, and "regenerative" guts, in which EBs are clustered and new ECs are prevalent. Using this system we first show that RNAi knockdown of Septate Junction proteins disrupts normal EB morphology and spatial distribution. With time-lapse imaging, we have also established that EBs are motile in nature, and when artificial tissue damage was introduced, exhibited increased cytoplasmic movements, and formed distinct clusters. We then demonstrate the utility of our pipeline in a small candidate screen for genes that might mediate EB clustering. Based on our results, we propose a working model that links the dynamic behaviour of EBs with midgut regeneration.

Cell signaling pathways involved in epithelial wound healing, show a lot of complexities when it comes to their regulation. Remarkably, a large proportion of these signaling pathways are triggered at the time of morphogenetic events which usually involve epithelial sheet fusions during embryonic development, such as the event of dorsal cloure in Drosophila embryos. One such conserved pathway in the wound healing process is the JNK-Dpp signaling pathway. Recent observations suggest that one such upstream regulator of JNK mediated apoptosis could be Rab11, a small Ras like GTPase, which is functionally associated with the membrane and cortical cytoskeletal organization of epithelial cells. Using Drosophila embryonic dorsal closure as a model of wound healing, we observed that a targeted expression of a Rab11 loss of function mutant in the dorso-lateral epidermis of fly embryos (tissue which extends contra-laterally in order to fill the intervening gap) undergoing dorsal closure leads to an ectopic expression of Caspase-3 and a concomitant up-regulation of the JNK-Dpp signaling. This resulted in the death of the dorso-lateral epithelial cells with a consequent embryonic lethality due to dorsal closure defects. Interestingly, a simultaneous knockdown of wingless (another developmentally conserved gene) in Rab11 mutants resulted in a rescue of the lethal phenotype and also a significant level of successful completion of the dorsal closure process. In our experiments we suggest Rab11 could promote cross talk between the JNK-Dpp pathway and the canonical wingless pathway in the regulation of apoptosis in the dorsolateral epithelium of fly embryos undergoing dorsal closure. One Sentence SummaryRab11 functions through a conserved Wingless mediated JNK-Dpp pathway during embryonic dorsal closure.

BackgroundA small number of signaling pathways regulates development in most animals, yet we do not know where these pathways are deployed in embryos of many animal phyla. Filling such gaps can contribute to understanding how diverse body shapes arise from differential deployment of conserved signaling pathways. Here, we examined where conserved pathways are deployed in tardigrades, a panarthropod phylum with miniaturized, segmented bodies. ResultsWe used in situ mRNA detection in the tardigrade Hypsibius exemplaris to reveal expression patterns of FGF and BMP signaling pathway components during body segmentation and early mesoderm development. Among the patterns examined, we found an FGF ligand and receptor expressed near each other in segmentally iterated regions of ectoderm and endomesoderm, respectively. We also found a BMP ligand and antagonist expressed in dorsoventrally-restricted patterns in the lateral ectoderm. ConclusionsThe detected patterns suggested specific hypotheses for further research: possible FGF signaling between ectoderm and endomesoderm, and possible roles of BMP signaling in dorsal-ventral patterning of lateral ectoderm. We compared our results with published expression patterns for FGF and BMP pathways across panarthropods, to contribute to previous hypotheses for how the development of segments and mesoderm may have evolved in the emergence of this clade of diversly-shaped animals. Bullet PointsO_LIDouble mRNA detection in tardigrades revealed expression patterns of FGF and BMP pathway genes C_LIO_LIfgf8 was detected primarily in ectoderm but absent from internal germ layers, and C_LIO_LIfgfrl1 was detected in all layers and enriched in internal layers C_LIO_LIdpp was detected more dorsally than sog in lateral ectoderm C_LIO_LIfgf8 and dpp gene expression exhibit different segmentally iterated domains in ectoderm C_LIO_LIResults suggest specific hypotheses for roles for FGF signaling and BMP signaling in tardigrade development C_LI

BackgroundIn almost all metazoans examined to this respect, the axial patterning system based on canonical Wnt (cWnt) signaling operates throughout the course of development. In most metazoans, gastrulation is polar, and embryos develop morphological landmarks of axial polarity, such as blastopore under control/regulation from Wnt signaling. However, in many cnidarian species, gastrulation is morphologically apolar. The question remains whether [c]Wnt signaling providing the establishment of a body axis controls morphogenetic processes involved in apolar gastrulation. ResultsIn this study, we focused on the embryonic development of Dynamena pumila, a cnidarian species with apolar gastrulation. We thoroughly described cell behavior, proliferation, and ultrastructure and examined axial patterning in the embryos of this species. We revealed that the first signs of morphological polarity appear only after the end of gastrulation, while molecular prepatterning of the embryo does exist during gastrulation. We have shown experimentally that in D. pumila, the morphological axis is highly robust against perturbations in cWnt activity. ConclusionOur results suggest that morphogenetic processes are uncoupled from molecular axial patterning during gastrulation in D. pumila. Investigation of D. pumila might significantly expand our understanding of the ways in which morphological polarization and axial molecular patterning are linked in Metazoa.

The Wnt1-Cre2 driver, designed to address the effect of Wnt1 overactivation in the ventral neural tube in the original Wnt1-Cre line, was recently shown to have ectopic expression in the male germline. When crossed with a reporter mouse, we observed fluorescent protein expression in non-neural-crest cell types in the gut. Here, we characterize the pattern of Cre-mediated recombination in the Wnt1-Cre2 driver using three transgenic reporter lines. We find aberrant reporter activation in the gut endoderm in embryonic and postnatal timepoints, starting as early as E8.5. This pattern of recombination was independent of the age, sex, and type of reporter line used, with the Wnt1-Cre2 allele inherited from either sires or dams resulting in ectopic fluorescence in the intestinal epithelium. We also detect reporter activity in the ventral neural tube. However, expression in the neural crest and its derivatives remained consistent with previous studies. We further quantify differences in the non-specific recombination observed across reporter lines using flow cytometry. Interestingly, the penetrance of reporter activation between reporter lines was different, with R26RmTmG showing less ectopic activation than the R26RtdTom and R26ReYFP lines. Finally, we propose a potential mechanism whereby genes surrounding the Wnt1-Cre2 insertion site on mouse chromosome 2 contribute to its Wnt1-independent activation in the endoderm. Taken together, our results suggest that users should exercise caution when using the Wnt1-Cre2 driver line for neural crest studies in the mouse.

Tuberin is a member of a large protein complex, Tuberous Sclerosis Complex, and acts as a sensor for nutrient status regulating protein synthesis and cell cycle progression. Mutations in the Tuberin gene, TSC2, lead to the formation of tumors and developmental defects in many organ systems, including the central nervous system. Tuberin is expressed in the brain throughout development and levels of Tuberin have been found to decrease during neuronal differentiation in cell lines in vitro. Our current work investigates the levels of Tuberin at two stages of embryonic development in vivo, and we study the mRNA and protein levels during a time course using immortalized cell lines in vitro. Our results show that Tuberin levels remain stable in the olfactory bulb but decrease in the Purkinje cell layer during embryonic mouse brain development. We show here that Tuberin levels are higher when cells are cultured as neurospheres, and knockdown of Tuberin results in a reduction in the number of neurospheres. These data provide support for the hypothesis that Tuberin is an important regulator of stemness and the reduction of Tuberin levels might support functional differentiation in the central nervous system. Understanding how Tuberin expression is regulated throughout neural development is essential to fully comprehend the role of this protein in several developmental and neural pathologies. HIGHLIGHTSO_LITuberin protein levels are decreased in the Purkinje cell layer in later stages of embryonic development. C_LIO_LITuberin protein and mRNA levels decrease as cells undergo neuronal differentiation. C_LIO_LIDownregulation of Tuberin impairs neurosphere formation. C_LIO_LITuberin is implicated in the maintenance of stemness in the developing brain. C_LI

Epithelial cells have a polarised distribution of protein complexes on the lateral membrane and are present as a polygonal array dominated by hexagons. Metazoan embryogenesis enables the study of temporal formation of the polygonal array and mechanisms that regulate its distribution. The plasma membrane of the syncytial Drosophila blastoderm embryo is organized as a polygonal array during cortical division cycles with an apical membrane and lateral furrow in between adjacent nuclei. We find that polygonal plasma membrane organization arises in syncytial division cycle 11 and hexagon dominance occurs with increase in furrow length in cycle 12. This is coincident with DE-cadherin and Bazooka enrichment at edges and the septin, Peanut enrichment at vertices of the base of the furrow. DE-cadherin depletion leads to loss of hexagon dominance. Bazooka and Peanut depletion leads to a delay in occurrence of hexagon dominance from nuclear cycle 12 to 13. Hexagon dominance in Bazooka and Peanut mutants occurs with furrow extension and correlates with increase in DE-cadherin in syncytial cycle 13. We conclude that a change in polarity complex distribution leads to loss of furrow stability thereby changing the polygonal organization of the blastoderm embryo.\n\nHighlight Summary for TOCMetazoan embryogenesis starts with the formation of polygonal epithelial-like cells. We show that hexagon dominance in polygonal epithelial-like plasma membrane organization occurs in nuclear cycle 12 in the syncytial blastoderm Drosophila embryo. DE-cadherin and Bazooka distribution along the lateral furrow regulates this hexagon dominance.

BackgroundLeft-right asymmetries are a common feature of metazoan nervous systems. This is particularly pronounced in the comparatively simple larval central nervous system (CNS) of the tunicate Ciona, whose swimming tadpole larva shows a clear chordate ground plan. While common pathway elements for specifying the left-right axis are found in the chordates, particularly a requirement for Nodal signaling, Ciona differs from its vertebrate cousins by specifying its axis at the neurula stage, rather than at gastrula. Additionally, Ciona, and other ascidians, have a requirement for an intact chorionic membrane for proper left/right specification. ResultsWe present here results showing that left-right asymmetry disruptions caused by removal of the chorion (dechorionation) are highly variable and present throughout the Ciona larval nervous system. While previous studies have documented disruptions to the conspicuously asymmetric sensory systems in the anterior brain vesicle, we document asymmetries in seemingly symmetric structures such as the posterior brain vesicle and motor ganglion. Moreover, defects caused by dechorionation include misplaced or absent neuron classes, loss of asymmetric gene expression, aberrant synaptic connectivity, and abnormal behaviors. In the motor ganglion, a brain structure that has been equated with the vertebrate hindbrain, we find that despite the apparent left/right symmetric distribution of interneurons and motor neurons, AMPA receptors are expressed exclusively on the left side, which equates with asymmetric swimming behaviors. We also find that within a population of dechorionated larvae, there is a small percentage with apparently normal left-right specification, and approximately equal population with inverted (mirror-image) asymmetry. We present a method based on a behavioral assay for isolating these larvae. When these two classes of larvae (normal and inverted) are assessed in a light dimming assay they display mirror-image behaviors, with normal larvae responding with counterclockwise swims, while inverted larvae respond with clockwise swims. ConclusionsOur findings highlight the importance of left-right specification pathways not only for proper CNS anatomy, but also for correct synaptic connectivity and behavior.

Energy storage and endocrine functions of the Drosophila fat body make it an excellent model for elucidating mechanisms that underlie physiological and pathophysiological organismal metabolism. Combined with Drosophilas robust genetic and immunofluorescence microscopy toolkits, studies of Drosophila fat body function are ripe for cell biological analysis. Unlike the larval fat body, which is easily removed as a single, cohesive sheet of tissue, isolating intact adult fat body proves to be more challenging, thus hindering consistent immunofluorescence labeling even within a single piece of adipose tissue. Here, we describe an improved approach to handling Drosophila abdomens that ensures full access of the adult fat body to solutions generally used in immunofluorescence labeling protocols. In addition, we assess the quality of fluorescence reporter expression and antibody immunoreactivity in response to variations in fixative type, fixation incubation time, and detergent used for cellular permeabilization. Overall, we provide several recommendations for steps in a whole mount staining protocol that results in consistent and robust immunofluorescence labeling of the adult Drosophila fat body. SUMMARY STATEMENTOptimization of adult Drosophila fat body fluorescence microscopy via modifications of tissue handling, fixation, and permeabilization steps in a whole mount immunolabeling protocol.

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of Mll3 and Mll4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these sister genes in mammalian development have only been incompletely reported. Here we examined the null phenotypes to establish that MLL3 is first required for lung maturation whereas MLL4 is first required for migration of the anterior visceral endoderm (AVE) that initiates gastrulation and is the first collective cell migration in development. This migration is preceded by a columnar to squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these sister genes argues against their action as general enhancer factors. Summary statementThe H3K4 methyltransferases MLL3 and MLL4 have strikingly different null phenotypes during mouse development; MLL3 is required for lung maturation whereas MLL4 is required for anterior visceral endoderm migration.

OverviewTo examine the hybrid hypothesis of human origins, a novel data mining program, BOOMSTICK, was used to scan the euchromatic portions of two target genomes, those of Homo sapiens and Pan paniscus. Each of the two genomes were broken up into 100-kB segments, each of which was searched for matches to a large set of porcine queries. All scans sought matches to the same set of 813,194 40-mer nucleotide queries randomly selected from the genome of Sus scrofa (domestic pig). For each of the two study organisms, mean segmental match rates (MSMRs) were then calculated for all segments in each of three categories: those segments occurring on autosomes, those on the X chromosome, and those on the Y chromosome. ResultsIn scans of single-copy regions (euchromatin) in both their Y chromosomes and their autosomes, it was found that the number of matches to randomly selected porcine queries was higher in humans than in bonobos. When autosomes were compared, matches were 1.3% higher in humans than in bonobos. This figure is equal to the percentage of human autosomal nucleotide positions bearing nucleotides that match in pig but not in bonobo. Remarkably, it agrees with the percentage of autosomal nucleotides previously reported to differ in bonobos and humans. So, the results of this study indicate that essentially all the nucleotide positions that differ in humans and bonobos, are the same in humans and pigs. In addition, the number of matches to pig queries found on the human Y chromosome was 34.5% higher than on the bonobo Y, and 12.4% higher than on the chimpanzee Y (the chimpanzee figure may be the more reliable of the two, since the bonobo Y nucleotide sequence file scanned contained only unlocalized scaffolds). MSMRs for the human and bonobo X chromosomes did not significantly differ.

Cell recruitment is a process by which a differentiated cell induces neighboring cells to adopt its same cell fate. In Drosophila, cells expressing the wing selector gene, vestigial (vg), drive a feed-forward recruitment signal that propagates Vg expression suggesting that the Vg pattern is established as a wavefront throughout the wing pouch. However, the dynamics of Vg pattern formation does not meet this expectation. Here we show that an induction signal may drive Vg expression without Vg feedforward recruitment several cells away from the dorsal-ventral (DV) boundary. Particularly, when Vg expression is strongly inhibited either at the DV boundary or away from it, the activation of the vg Quadrant Enhancer still occurs at a distance, although the levels and precision of the pattern are compromised. Using live imaging of a dual-fluorescent reporter system, we show that contact-dependent cell recruitment does occur in wild-type conditions suggesting that a combination of a long-range induction signal and a signal-relay recruitment establishes Vg patterning in a robust manner. Overall, our findings reveal a previously unidentified role of cell recruitment as a robustness-conferring patterning mechanism. Summary StatementRobust formation of the Vg pattern is established by two mechanisms: a fast, long-range, but noisy induction signal; and a more precise signal relay, contact-dependent, cell recruitment process.

The Wnt/{beta}-catenin signaling pathway is highly conserved throughout evolution and it plays crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathways activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12x or 16x) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterisation of TCF/{beta}-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.