Labeling the Micropylar Cell in Zebrafish Whole-Mount and Cryo-sectioned Follicles.

In some animal species, fertilization occurs through a funnel-like canal called the "micropyle." In teleost fishes, the micropyle is formed by a very specialized follicle cell, called the micropylar cell (MC). Very little is known about the mechanisms underlying the specification and differentiation of the MC, a unique cell among hundreds that compose the follicle cell layer. The Hippo pathway effector Taz is essential for this process and is the first reported MC marker. Here, we describe a method to identify and mark the micropylar cell following the immunostaining procedure on cryosections or combining it with the RNA in situ hybridization on whole-mount follicles.

Morphogenesis is transcriptionally coupled to neurogenesis during peripheral olfactory organ development.

Sense organs acquire their distinctive shapes concomitantly with the differentiation of sensory cells and neurons necessary for their function. Although our understanding of the mechanisms controlling morphogenesis and neurogenesis in these structures has grown, how these processes are coordinated remains largely unexplored. Neurogenesis in the zebrafish olfactory epithelium requires the bHLH proneural transcription factor Neurogenin 1 (Neurog1). To address whether Neurog1 also controls morphogenesis, we analysed the migratory behaviour of early olfactory neural progenitors in neurog1 mutant embryos. Our results indicate that the oriented movements of these progenitors are disrupted in this context. Morphogenesis is similarly affected by mutations in the chemokine receptor gene, cxcr4b, suggesting it is a potential Neurog1 target gene. We find that Neurog1 directly regulates cxcr4b through an E-box cluster located just upstream of the cxcr4b transcription start site. Our results suggest that proneural transcription factors, such as Neurog1, directly couple distinct aspects of nervous system development.

Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis.

Cilia and the intraflagellar transport (IFT) proteins involved in ciliogenesis are associated with congenital heart diseases (CHDs). However, the molecular links between cilia, IFT proteins, and cardiogenesis are yet to be established. Using a combination of biochemistry, genetics, and live-imaging methods, we show that IFT complex B proteins (Ift88, Ift54, and Ift20) modulate the Hippo pathway effector YAP1 in zebrafish and mouse. We demonstrate that this interaction is key to restrict the formation of the proepicardium and the myocardium. In cellulo experiments suggest that IFT88 and IFT20 interact with YAP1 in the cytoplasm and functionally modulate its activity, identifying a molecular link between cilia-related proteins and the Hippo pathway. Taken together, our results highlight a noncanonical role for IFT complex B proteins during cardiogenesis and shed light on a mechanism of action for ciliary proteins in YAP1 regulation.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Standardized mounting method of (zebrafish) embryos using a 3D-printed stamp for high-content, semi-automated confocal imaging.

BACKGROUND: Developmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation. RESULTS: Here we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple µ-wells in an agarose cast. Each µ-well models a negative of the average zebrafish embryo morphology between 22 and 96 h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR). CONCLUSIONS: We present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of µ-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.

Biomechanical signaling within the developing zebrafish heart attunes endocardial growth to myocardial chamber dimensions.

Intra-organ communication guides morphogenetic processes that are essential for an organ to carry out complex physiological functions. In the heart, the growth of the myocardium is tightly coupled to that of the endocardium, a specialized endothelial tissue that lines its interior. Several molecular pathways have been implicated in the communication between these tissues including secreted factors, components of the extracellular matrix, or proteins involved in cell-cell communication. Yet, it is unknown how the growth of the endocardium is coordinated with that of the myocardium. Here, we show that an increased expansion of the myocardial atrial chamber volume generates higher junctional forces within endocardial cells. This leads to biomechanical signaling involving VE-cadherin, triggering nuclear localization of the Hippo pathway transcriptional regulator Yap1 and endocardial proliferation. Our work suggests that the growth of the endocardium results from myocardial chamber volume expansion and ends when the tension on the tissue is relaxed.

Collective cell migration: general themes and new paradigms.

Collective cell migration plays essential roles in embryogenesis and also contributes to disease states. Recent years have seen immense progress in understanding mechanisms and overarching concepts of collective cell migration. Self-organization of moving groups emerges as an important common feature. This includes self-generating gradients, internal chemotaxis or mechanotaxis and contact-dependent polarization within migrating cell groups. Here, we will discuss these concepts and their applications to classical models of collective cell migration. Further, we discuss new models and paradigms of collective cell migration and elaborate on open questions and future challenges. Answering these questions will help to expand our appreciation of this exciting theme in developmental cell biology and contribute to the understanding of disease states.

Yap/Taz-TEAD activity links mechanical cues to progenitor cell behavior during zebrafish hindbrain segmentation.

Cells perceive their microenvironment through chemical and physical cues. However, how the mechanical signals are interpreted during embryonic tissue deformation to result in specific cell behaviors is largely unknown. The Yap/Taz family of transcriptional co-activators has emerged as an important regulator of tissue growth and regeneration, responding to physical cues from the extracellular matrix, and to cell shape and actomyosin cytoskeletal changes. In this study, we demonstrate the role of Yap/Taz-TEAD activity as a sensor of mechanical signals in the regulation of the progenitor behavior of boundary cells during zebrafish hindbrain compartmentalization. Monitoring of in vivo Yap/Taz activity during hindbrain segmentation indicated that boundary cells responded to mechanical cues in a cell-autonomous manner through Yap/Taz-TEAD activity. Cell-lineage analysis revealed that Yap/Taz-TEAD boundary cells decreased their proliferative activity when Yap/Taz-TEAD activity ceased, which preceded changes in their cell fate from proliferating progenitors to differentiated neurons. Functional experiments demonstrated the pivotal role of Yap/Taz-TEAD signaling in maintaining progenitor features in the hindbrain boundary cell population.

The Hippo pathway effector Taz is required for cell morphogenesis and fertilization in zebrafish.

Hippo signaling is a critical pathway that integrates extrinsic and intrinsic mechanical cues to regulate organ size. Despite its essential role in organogenesis, little is known about its role in cell fate specification and differentiation. Here, we unravel a novel and unexpected role of the Hippo pathway effector Taz (wwtr1) in controlling the size, shape and fate of a unique cell in the zebrafish ovary. We show that wwtr1 mutant females are infertile. In teleosts, fertilization occurs through the micropyle, a funnel-like opening in the chorion, formed by a unique enlarged follicle cell, the micropylar cell (MC). We describe here, for the first time, the mechanism that underlies the differentiation of the MC. Our genetic analyses show that Taz is essential for MC fate acquisition and subsequent micropyle formation in zebrafish. We identify Taz as the first bona fide MC marker and show that Taz is specifically and strongly enriched in the MC precursor. Altogether, we performed the first genetic and molecular characterization of the MC and propose that Taz is a key regulator of MC fate.This article has an associated 'The people behind the papers' interview.

CXCL12 and MYC control energy metabolism to support adaptive responses after kidney injury.

Kidney injury is a common complication of severe disease. Here, we report that injuries of the zebrafish embryonal kidney are rapidly repaired by a migratory response in 2-, but not in 1-day-old embryos. Gene expression profiles between these two developmental stages identify cxcl12a and myca as candidates involved in the repair process. Zebrafish embryos with cxcl12a, cxcr4b, or myca deficiency display repair abnormalities, confirming their role in response to injury. In mice with a kidney-specific knockout, Cxcl12 and Myc gene deletions suppress mitochondrial metabolism and glycolysis, and delay the recovery after ischemia/reperfusion injury. Probing these observations in zebrafish reveal that inhibition of glycolysis slows fast migrating cells and delays the repair after injury, but does not affect the slow cell movements during kidney development. Our findings demonstrate that Cxcl12 and Myc facilitate glycolysis to promote fast migratory responses during development and repair, and potentially also during tumor invasion and metastasis.

Progesterone modulates microtubule dynamics and epiboly progression during zebrafish gastrulation.

Control of microtubule dynamics is crucial for cell migration. We analyzed regulation of microtubule network dynamics in the zebrafish yolk cell during epiboly, the earliest coordinated gastrulation movement. We labeled microtubules with EMTB-3GFP and EB3-mCherry to visualize and measure microtubule dynamics by TIRF microscopy live imaging. Yolk cell microtubules dynamics is temporally modulated during epiboly progression. We used maternal zygotic Pou5f3 mutant (MZspg) embryos, which develop strong distortions of microtubule network organization and epiboly retardation, to investigate genetic control of microtubule dynamics. In MZspg embryos, microtubule plus-end growth tracks move slower and are less straight compared to wild-type. MZspg embryos have altered steroidogenic enzyme expression, resulting in increased pregnenolone and reduced progesterone levels. We show that progesterone positively affects microtubule plus-end growth and track straightness. Progesterone may thus act as a non-cell-autonomous regulator of microtubule dynamics across the large yolk cell, and may adjust differing demands on microtubule dynamics and stability during initiation and progression phases of epiboly.

Flow-Dependent Endothelial YAP Regulation Contributes to Vessel Maintenance.

Endothelial cells (ECs) line the inside of blood vessels and respond to mechanical cues generated by blood flow. Mechanical stimuli regulate the localization of YAP by reorganizing the actin cytoskeleton. Here we demonstrate blood-flow-mediated regulation of endothelial YAP in vivo. We indirectly monitored transcriptional activity of Yap1 (zebrafish YAP) and its spatiotemporal localization in living zebrafish and found that Yap1 entered the nucleus and promoted transcription in response to blood flow. In cultured human ECs, laminar shear stress induced nuclear import of YAP and its transcriptional activity in a manner independent of Hippo signaling. We uncovered a molecular mechanism by which flow induced the nuclear translocation of YAP through the regulation of filamentous actin and angiomotin. Yap1 mutant zebrafish showed a defect in vascular stability, indicating an essential role for Yap1 in blood vessels. Our data imply that endothelial Yap1 functions in response to flow to maintain blood vessels.

Amotl2a interacts with the Hippo effector Yap1 and the Wnt/ß-catenin effector Lef1 to control tissue size in zebrafish.

During development, proliferation must be tightly controlled for organs to reach their appropriate size. While the Hippo signaling pathway plays a major role in organ growth control, how it senses and responds to increased cell density is still unclear. In this study, we use the zebrafish lateral line primordium (LLP), a group of migrating epithelial cells that form sensory organs, to understand how tissue growth is controlled during organ formation. Loss of the cell junction-associated Motin protein Amotl2a leads to overproliferation and bigger LLP, affecting the final pattern of sensory organs. Amotl2a function in the LLP is mediated together by the Hippo pathway effector Yap1 and the Wnt/ß-catenin effector Lef1. Our results implicate for the first time the Hippo pathway in size regulation in the LL system. We further provide evidence that the Hippo/Motin interaction is essential to limit tissue size during development.

Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation.

The directed migration of cell collectives drives the formation of complex organ systems. A characteristic feature of many migrating collectives is a 'tissue-scale' polarity, whereby 'leader' cells at the edge of the tissue guide trailing 'followers' that become assembled into polarised epithelial tissues en route. Here, we combine quantitative imaging and perturbation approaches to investigate epithelial cell state transitions during collective migration and organogenesis, using the zebrafish lateral line primordium as an in vivo model. A readout of three-dimensional cell polarity, based on centrosomal-nucleus axes, allows the transition from migrating leaders to assembled followers to be quantitatively resolved for the first time in vivo. Using live reporters and a novel fluorescent protein timer approach, we investigate changes in cell-cell adhesion underlying this transition by monitoring cadherin receptor localisation and stability. This reveals that while cadherin 2 is expressed across the entire tissue, functional apical junctions are first assembled in the transition zone and become progressively more stable across the leader-follower axis of the tissue. Perturbation experiments demonstrate that the formation of these apical adherens junctions requires dynamic microtubules. However, once stabilised, adherens junction maintenance is microtubule independent. Combined, these data identify a mechanism for regulating leader-to-follower transitions within migrating collectives, based on the relocation and stabilisation of cadherins, and reveal a key role for dynamic microtubules in this process.

Shroom3 is required downstream of FGF signalling to mediate proneuromast assembly in zebrafish.

During development, morphogenetic processes require a precise coordination of cell differentiation, cell shape changes and, often, cell migration. Yet, how pattern information is used to orchestrate these different processes is still unclear. During lateral line (LL) morphogenesis, a group of cells simultaneously migrate and assemble radially organized cell clusters, termed rosettes, that prefigure LL sensory organs. This process is controlled by Fibroblast growth factor (FGF) signalling, which induces cell fate changes, cell migration and cell shape changes. However, the exact molecular mechanisms induced by FGF activation that mediate these changes on a cellular level are not known. Here, we focus on the mechanisms by which FGFs control apical constriction and rosette assembly. We show that apical constriction in the LL primordium requires the activity of non-muscle myosin. We demonstrate further that shroom3, a well-known regulator of non-muscle myosin activity, is expressed in the LL primordium and that its expression requires FGF signalling. Using gain- and loss-of-function experiments, we demonstrate that Shroom3 is the main organizer of cell shape changes during rosette assembly, probably by coordinating Rho kinase recruitment and non-muscle myosin activation. In order to quantify morphogenesis in the LL primordium in an unbiased manner, we developed a unique trainable 'rosette detector'. We thus propose a model in which Shroom3 drives rosette assembly in the LL downstream of FGF in a Rho kinase- and non-muscle myosin-dependent manner. In conclusion, we uncovered the first mechanistic link between patterning and morphogenesis during LL sensory organ formation.

Fgf and Sdf-1 pathways interact during zebrafish fin regeneration.

The chemokine stromal cell-derived factor-1 (SDF1) was originally identified as a pre-B cell stimulatory factor but has been recently implicated in several other key steps in differentiation and morphogenesis. In addition, SDF1 as well as FGF signalling pathways have recently been shown to be involved in the control of epimorphic regeneration. In this report, we address the question of a possible interaction between the two signalling pathways during adult fin regeneration in zebrafish. Using a combination of pharmaceutical and genetic tools, we show that during epimorphic regeneration, expression of sdf1, as well as of its cognate receptors, cxcr4a, cxcr4b and cxcr7 are controlled by FGF signalling. We further show that, Sdf1a negatively regulates the expression of fgf20a. Together, these results lead us to propose that: 1) the function of Fgf in blastema formation is, at least in part, relayed by the chemokine Sdf1a, and that 2) Sdf1 exerts negative feedback on the Fgf pathway, which contributes to a transient expression of Fgf20a downstream genes at the beginning of regeneration. However this feedback control can be bypassed since the Sdf1 null mutants regenerate their fin, though slower. Very few mutants for the regeneration process were isolated so far, illustrating the difficulty in identifying genes that are indispensable for regeneration. This observation supports the idea that the regeneration process involves a delicate balance between multiple pathways.

Control of convergent yolk syncytial layer nuclear movement in zebrafish.

Nuclear movements play an essential role in metazoan development. Although the intracellular transport mechanisms underlying nuclear movements have been studied in detail, relatively little is known about signals from surrounding cells and tissues controlling these movements. Here, we show that, in gastrulating zebrafish embryos, convergence movements of nuclei within the yolk syncytial layer (YSL) are guided by mesoderm and endoderm progenitors migrating along the surface of the yolk towards the dorsal side of the developing gastrula. Progenitor cells direct the convergence movements of internal yolk syncytial nuclei (iYSN) by modulating cortical flow within the YSL in which the iYSN are entrained. The effect of mesoderm and endoderm progenitors on the convergence movement of iYSN depends on the expression of E-cadherin, indicating that adhesive contact between the cells and the YSL is required for the mesendoderm-modulated YSL cortical flow mediating nuclear convergence. In summary, our data reveal a crucial function for cortical flow in the coordination of syncytial nuclear movements with surrounding cells and tissues during zebrafish gastrulation.

A functional interaction between Irx and Meis patterns the anterior hindbrain and activates krox20 expression in rhombomere 3.

Patterning of the vertebrate hindbrain involves a segmentation process leading to the formation of seven rhombomeres along the antero-posterior axis. While recent studies have shed light on the mechanisms underlying progressive subdivision of the posterior hindbrain into individual rhombomeres, the early events involved in anterior hindbrain patterning are still largely unknown. In this paper we demonstrate that two zebrafish Iroquois transcription factors, Irx7 and Irx1b, are required for the proper formation and specification of rhombomeres 1 to 4 and, in particular, for krox20 activation in r3. We also show that Irx7 functionally interacts with Meis factors to activate the expression of anterior hindbrain markers, such as hoxb1a, hoxa2 and krox20, ectopically in the anterior neural plate. Then, focusing on krox20 expression, we show that the effect of Irx7 and Meis1.1 is mediated by element C, a conserved cis-regulatory element involved in krox20 activation in the hindbrain. Together, our data point to an essential function of Iroquois transcription factors in krox20 activation and, more generally, in anterior hindbrain specification.

Dynamic Fgf signaling couples morphogenesis and migration in the zebrafish lateral line primordium.

The collective migration of cells in the form of cohesive tissues is a hallmark of both morphogenesis and repair. The extrinsic cues that direct these complex migrations usually act by regulating the dynamics of a specific subset of cells, those at the leading edge. Given that normally the function of tissue migration is to lay down multicellular structures, such as branched epithelial networks or sensory organs, it is surprising how little is known about the mechanisms that organize cells behind the leading edge. Cells of the zebrafish lateral line primordium switch from mesenchyme-like leader cells to epithelial rosettes that develop into mechanosensory organs. Here, we show that this transition is regulated by an Fgf signaling circuit that is active within the migrating primordium. Point sources of Fgf ligand drive surrounding cells towards a ;non-leader' fate by increasing their epithelial character, a prerequisite for rosette formation. We demonstrate that the dynamic expression of Fgf ligands determines the spatiotemporal pattern of epithelialization underlying sensory organ formation in the lateral line. Furthermore, this work uncovers a surprising link between internal tissue organization and collective migration.

In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum.

Although cells migrate in a constrained 3D environment in vivo, in-vitro studies have mainly focused on the analysis of cells moving on 2D substrates. Under such conditions, the Golgi complex is always located towards the leading edge of the cell, suggesting that it is involved in the directional movement. However, several lines of evidence indicate that this location can vary depending on the cell type, the environment or the developmental processes. We have used micro contact printing (microCP) to study the migration of cells that have a geometrically constrained shape within a polarized phenotype. Cells migrating on micropatterned lines of fibronectin are polarized and migrate in the same direction. Under such conditions, the Golgi complex and the centrosome are located behind the nucleus. In addition, the Golgi complex is often displaced several micrometres away from the nucleus. Finally, we used the zebrafish lateral line primordium as an in-vivo model of cells migrating in a constrained environment and observe a similar localization of both the Golgi and the centrosome in the leading cells. We propose that the positioning of the Golgi complex and the centrosome depends on the geometrical constraints applied to the cell rather than on a precise migratory function in the leading region.