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STUDY QUESTION Is the vertebrate protein Dead end (DND1) a causative factor for human infertility and can novel in vivo assays in zebrafish help in evaluating this? SUMMARY ANSWER Combining patient genetic data with functional in vivo assays in zebrafish reveals a possible role for DND1 in human male fertility. WHAT IS KNOWN ALREADY About 7% of the male population is affected by infertility but linking specific gene variants to the disease is challenging. The function of the DND1 protein was shown to be critical for germ cell development in several model organisms but a reliable and cost-effective method for evaluating the activity of the protein in the context of human male infertility is still missing. STUDY DESIGN, SIZE, DURATION Exome data from 1305 men included in the Male Reproductive Genomics cohort were examined in this study. A total of 1114 of the patients showed severely impaired spermatogenesis but were otherwise healthy. Eighty-five men with intact spermatogenesis were included in the study as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS We screened the human exome data for rare, stop-gain, frameshift, splice site, as well as missense variants in DND1. The results were validated by Sanger sequencing. Immunohistochemical techniques and, when possible, segregation analyses were performed for patients with identified DND1 variants. The amino acid exchange in the human variant was mimicked at the corresponding site of the zebrafish protein. Using different aspects of germline development in live zebrafish embryos as biological assays, we examined the activity level of these DND1 protein variants. MAIN RESULTS AND THE ROLE OF CHANCE In human exome sequencing data, we identified four heterozygous variants in DND1 (three missense and one frameshift variant) in five unrelated patients. The function of all of the variants was examined in the zebrafish and one of those was studied in more depth in this model. We demonstrate the use of zebrafish assays as a rapid and effective biological readout for evaluating the possible impact of multiple gene variants on male fertility. This in vivo approach allowed us to assess the direct impact of the variants on germ cell function in the context of the native germline. Focusing on the DND1 gene, we find that zebrafish germ cells, expressing orthologs of DND1 variants identified in infertile men, failed to arrive correctly at the position where the gonad develops and exhibited defects in cell fate maintenance. Importantly, our analysis facilitated the evaluation of single nucleotide variants, whose impact on protein function is difficult to predict, and allowed us to distinguish variants that do not affect the protein’s activity from those that strongly reduce it and could thus potentially be the primary cause for the pathological condition. These aberrations in germline development resemble the testicular phenotype of azoospermic patients. LIMITATIONS, REASONS FOR CAUTION The pipeline we present requires access to zebrafish embryos and to basic imaging equipment. The notion that the activity of the protein in the zebrafish-based assays is relevant for the human homolog is well supported by previous knowledge. Nevertheless, the human protein may differ in some respects from its homologue in zebrafish. Thus, the assay should be considered only one of the parameters used in defining DND1 variants as causative or non-causative for infertility. WIDER IMPLICATIONS OF THE FINDINGS Using DND1 as an example, we have shown that the approach described in this study, relying on bridging between clinical findings and fundamental cell biology, can help to establish links between novel human disease candidate genes and fertility. In particular, the power of the approach we developed is manifested by the fact that it allows the identification of DND1 variants that arose de novo. The strategy presented here can be applied to different genes in other disease contexts. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the German Research Foundation, Clinical Research Unit, CRU326 ‘Male Germ Cells’. There are no competing interests. TRIAL REGISTRATION NUMBER N/A.

In both physiological processes and disease contexts, migrating cells have the ability to adapt to conditions in their environment. As an in vivo model for this process, we use zebrafish primordial germ cells that migrate throughout the developing embryo. When migrating within an ectodermal environment, the germ cells form fewer and smaller blebs when compared with their behavior within mesodermal environment. We find that cortical tension of neighboring cells is a parameter that affects blebbing frequency. Interestingly, the change in blebbing activity is accompanied by the formation of more actin-rich protrusions. These alterations in cell behavior that correlate with changes in RhoA activity could allow the cells to maintain dynamic motility parameters, such as migration speed and track straightness, in different settings. In addition, we find that the polarity of the cells can be affected by stiff structures positioned in their migration path This article has an associated ‘The people behind the papers’ interview.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvβ5 integrin. JAM-A binds Csk and inhibits the activity of αvβ5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell–cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell–matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvβ5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

SUMMARYGerm granules, condensates of phase-separated RNA and protein, are essential for germline development, but how these molecules are organized within the granules and whether such an organization is relevant for germ cell fate is unclear. Combining three-dimensionalin vivostructural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the vertebrate-specific Dead end protein is essential for positioningnanos3RNA at the condensates’ periphery, where ribosomes are located. Without Dead end, or when translation is inhibited,nanos3RNA translocates into granule interiors, far from the ribosomes’ location. These findings reveal the molecular mechanisms controlling the spatial organization of RNA within the phase-separated organelle and the importance of subgranule RNA localization for preserving germ cell totipotency.

Cell ablation is a key method in the research fields of developmental biology, tissue regeneration, and tissue homeostasis. Eliminating specific cell populations allows for characterizing interactions that control cell differentiation, death, behavior, and spatial organization of cells. Current methodologies for inducing cell death suffer from relatively slow kinetics, making them unsuitable for analyzing rapid events and following primary and immediate consequences of the ablation. To address this, we developed a cell-ablation system that is based on bacterial toxin/anti-toxin proteins and enables rapid and cell-autonomous elimination of specific cell types and organs in zebrafish embryos. A unique feature of this system is that it uses an anti-toxin, which allows for controlling the degree and timing of ablation and the resulting phenotypes. The transgenic zebrafish generated in this work represent a highly efficient tool for cell ablation, and this approach is applicable to other model organisms as demonstrated here for Drosophila.

Zebrafish primordial germ cells (PGCs) constitute a useful in vivo model to study cell migration and to elucidate the role of specific proteins in this process. Here we report on the role of the heat shock protein Hsp90aa1.2, a protein whose RNA level is elevated in the PGCs during their migration. Reducing Hsp90aa1.2 activity slows down the progression through the cell cycle and leads to defects in the control over the MTOC number in the migrating cells. These defects result in a slower migration rate and compromise the arrival of PGCs at their target, the region where the gonad develops. Our results emphasize the importance of ensuring rapid progression through the cell cycle during single-cell migration and highlight the role of heat shock proteins in the process.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis., The formation of new blood vessels requires both polarized cell migration and coordinated control of endothelial cell contacts. Here, Cao and colleagues describe at the sub-cellular level the cytoskeletal and cell junction dynamics regulating these processes upon VEGF-induced cell elongation.

Two waves of DNA methylation reprogramming occur during mammalian embryogenesis; during preimplantation development and during primordial germ cell (PGC) formation. However, it is currently unclear how evolutionarily conserved these processes are. Here we characterise the DNA methylomes of zebrafish PGCs at four developmental stages and identify retention of paternal epigenetic memory, in stark contrast to the findings in mammals. Gene expression profiling of zebrafish PGCs at the same developmental stages revealed that the embryonic germline is defined by a small number of markers that display strong developmental stage-specificity and that are independent of DNA methylation-mediated regulation. We identified promoters that are specifically targeted by DNA methylation in somatic and germline tissues during vertebrate embryogenesis and that are frequently misregulated in human cancers. Together, these detailed methylome and transcriptome maps of the zebrafish germline provide insight into vertebrate DNA methylation reprogramming and enhance our understanding of the relationships between germline fate acquisition and oncogenesis., Germ cells are the means of transferring genetic information to the next generation. Here the authors characterise the DNA methylomes of zebrafish primordial germ cells and find that, unlike mammals, the zebrafish germ cells do not undergo genome-wide DNA demethylation but rather retain paternal DNA methylation patterns

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.

Stem cell fate decisions are controlled by a molecular network in which transcription factors and miRNAs are of key importance. To systemically investigate their impact on neural stem cell (NSC) maintenance and neuronal commitment, we performed a high-throughput mRNA and miRNA profiling and isolated functional interaction networks of involved mechanisms. Thereby, we identified an E2F1–miRNA feedback loop as important regulator of NSC fate decisions. Although E2F1 supports NSC proliferation and represses transcription of miRNAs from the miR-17∼92 and miR-106a∼363 clusters, these miRNAs are transiently up-regulated at early stages of neuronal differentiation. In these early committed cells, increased miRNAs expression levels directly repress E2F1 mRNA levels and inhibit cellular proliferation. In mice, we demonstrated that these miRNAs are expressed in the neurogenic areas and that E2F1 inhibition represses NSC proliferation. The here presented data suggest a novel interaction mechanism between E2F1 and miR-17∼92 / miR-106a∼363 miRNAs in controlling NSC proliferation and neuronal differentiation. Fil: Palm, Thomas. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania Fil: Hemmer, Kathrin. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania Fil: Winter, Julia. Helmholtz-University-Group Molecular RNA Biology & Cancer. German Cancer Research Center; Alemania. University of Heidelberg. Institute of Pathology; Alemania Fil: Fricke, Inga B.. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania. Westfälische Wilhelms-Universität Münster. European Institute for Molecular Imaging; Alemania. Interdisciplinary Center for Clinical Research; Alemania Fil: Tarbashevich, Katsiaryna. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology; Alemania Fil: Sadeghi Shakib, Fereshteh. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania Fil: Rudolph, Ina-Maria. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania Fil: Hillje, Anna Lena. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania Fil: de Luca, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Bahnassawy, Lamia’a. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania Fil: Madel, Rabea. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania Fil: Viel, Thomas. Westfälische Wilhelms-Universität Münster. European Institute for Molecular Imaging; Alemania. Interdisciplinary Center for Clinical Research; Alemania Fil: de Siervi, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Jacobs, Andreas H.. Westfälische Wilhelms-Universität Münster. European Institute for Molecular Imaging; Alemania. Interdisciplinary Center for Clinical Research; Alemania Fil: Diederichs, Sven. Helmholtz-University-Group Molecular RNA Biology & Cancer. German Cancer Research Center; Alemania. University of Heidelberg. Institute of Pathology; Alemania Fil: Schwamborn, Jens C.. Westfälische Wilhelms-Universität Münster. Center for Molecular Biology of Inflammation. Institute of Cell Biology. Stem Cell Biology and Regeneration Group; Alemania. Interdisciplinary Center for Clinical Research; Alemania

Triple negative breast cancer (TNBC) is a highly aggressive and recurrent type of breast carcinoma that is associated with poor patient prognosis. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. The CXCR4-CXCL12 chemokine signaling axis guides cell migration in physiological and pathological processes including breast cancer metastases. Although targeted therapies to inhibit the CXCR4-CXCL12 axis are under clinical experimentation, still no effective therapeutic approaches have been established to block CXCR4 in TNBC. To unravel the role of the CXCR4-CXCL12 axis in TNBC early metastasis formation, we used the zebrafish xenograft model. Importantly, we demonstrate that cross communication between the zebrafish and human ligands and receptors takes place and human tumor cells expressing CXCR4 initiate early metastatic events by sensing zebrafish cognate ligands at the metastatic site. Taking advantage of the conserved intercommunication between human tumor cells and the zebrafish host, we blocked TNBC early metastatic events by chemical and genetic inhibition of CXCR4 signaling. We used IT1t, a potent CXCR4 antagonist, and show for the first time its promising anti-tumor effects. In conclusion, we confirm the validity of the zebrafish as a xenotransplantation model and propose a pharmacological approach to target CXCR4 in TNBC.

Grip2.1 is a conserved PDZ-domain protein with a function in the context of primordial germ cell development and migration in Xenopus embryos. Its mRNA is maternally supplied and found to be associated with the germ plasm, located at the tip of the vegetal cortex in Xenopus oocytes. Here, we demonstrate that the 3'-UTR of XGrip2.1 contains a 211 nucleotide RNA signal sequence that promotes localization to the mitochondrial cloud via the early localization pathway upon injection into stage I oocytes. The same element is also capable of using the late transport pathway if injected into stage III/IV oocytes. In vitro protein interaction studies reveal binding to ElrA/B, Vg1RBP and VgRBP60, proteins that have previously been associated with the vegetal localization machinery. Mutational interference with Vg1RBP and VgRBP60 binding severely reduces early and late localization activity. Selective interference with Vg1RBP binding significantly reduces late localization while having only a mild effect on localization to the mitochondrial cloud, indicating that the signal sequences and protein machinery required for early and late pathway localization though overlapping are not identical.

International audience; The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes.

Cell migration is essential for morphogenesis, for organ formation and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model to study this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility and with the ability of the cells to reach their target.

Maintaining cell fate relies on robust mechanisms that prevent the differentiation of specified cells into other cell types. This is especially critical during embryogenesis, when extensive cell proliferation, patterning, and migration events take place. Here we show that vertebrate primordial germ cells (PGCs) are protected from reprogramming into other cell types by the RNA-binding protein Dead end (Dnd). PGCs knocked down for Dnd lose their characteristic morphology and adopt various somatic cell fates. Concomitantly, they gain a gene expression profile reflecting differentiation into cells of different germ layers, in a process that we could direct by expression of specific cell-fate determinants. Importantly, we visualized these events within live zebrafish embryos, which provide temporal information regarding cell reprogramming. Our results shed light on the mechanisms controlling germ cell fate maintenance and are relevant for the formation of teratoma, a tumor class composed of cells from more than one germ layer.

Directional transport of specific mRNAs is of primary biological relevance. In Xenopus oocytes, mRNA localization to the vegetal pole is important for germ layer formation and germ cell development. Using a biochemical approach, we identified Xenopus Elr-type proteins, homologs of the Hu/ELAV proteins, as novel components of the vegetal mRNA localization machinery. They bind specifically to the localization elements of several different vegetally localizing Xenopus mRNAs, and they are part of one RNP together with other localization proteins, such as Vg1RBP and XStaufen 1. Blocking Elr-type protein binding by either localization element mutagenesis or antisense morpholino oligonucleotide-mediated masking of their target RNA structures, as well as overexpression of wild type and mutant ElrB proteins, interferes with vegetal localization in Xenopus oocytes.

Cell migration and polarization is controlled by signals in the environment. Migrating cells typically form filopodia that extend from the cell surface, but the precise function of these structures in cell polarization and guided migration is poorly understood. Using the in vivo model of zebrafish primordial germ cells for studying chemokine-directed single cell migration, we show that filopodia distribution and their dynamics are dictated by the gradient of the chemokine Cxcl12a. By specifically interfering with filopodia formation, we demonstrate for the first time that these protrusions play an important role in cell polarization by Cxcl12a, as manifested by elevation of intracellular pH and Rac1 activity at the cell front. The establishment of this polarity is at the basis of effective cell migration towards the target. Together, we show that filopodia allow the interpretation of the chemotactic gradient in vivo by directing single-cell polarization in response to the guidance cue. DOI: http://dx.doi.org/10.7554/eLife.05279.001, eLife digest Some of the cells in an animal embryo have to migrate long distances to reach their final positions; that is to say, to reach the locations where they will participate in the formation of tissues and organs. The migration of cells is also important throughout the entire lifespan of an animal. White blood cells, for example, must be able to move within tissues to search for and fight infections as well as to detect and remove abnormal cells. The front end of a migrating cell typically protrudes. The back of the cell is then pulled and detaches, which allows the whole cell to move forward. Migrating cells generate thin finger-like projections known as filopodia that have been suggested to help the cell sense their external environments and follow chemical cues. It is not clear what happens to a migrating cell in a living organism if the formation of its filopodia is impaired, or even how filipodia help the normal migration of cells in animals. To define how filopodia help to guide migrating cells in an animal, Meyen et al. analyzed the migration of cells called ‘primordial germ cells’ (or PGCs) in zebrafish. These cells form very early on in development of a zebrafish embryo at a position that is far away from their final location (in the testes or ovaries where they will go on to form sperm or egg cells respectively). Meyen et al. revealed that cells that are exposed to the guidance cue (a protein called a chemokine) form more filopodia at their front compared to their rear. The filopodia formed at the cell front also extend and retract more frequently. Meyen et al. further observed that the specific chemokine that guides the cells can bind to the filopodia and enter the cell. This leads to a signal inside the cell that tells the cell to move in the direction where more of the chemokine is found. Indeed, altering the distribution and number of filopodia around the cell's edge decreases the ability of the primordial germ cells to reach their targets. Together, this work shows that the filopodia at the front end of cells are required for sensing the chemokines that guide cell movement. Further work is required to understand the mechanism that determines the distribution of filopodia on the surface of migrating cells, and the role of chemokines in the process. Moreover, this work may also be relevant for understanding the migration of cancer cells, because several types of cancer can invade new tissues by following directional cues including chemokines. DOI: http://dx.doi.org/10.7554/eLife.05279.002

Significance Cell motility is critical for a wide range of processes in development, homeostasis, and immune response. Conversely, abnormal regulation of cell migration leads to pathological consequences like cancer metastasis. In this study, we investigate the mechanisms controlling the timing of motility acquisition, using zebrafish primordial germ cells as an in vivo model. We defined a previously unknown role for the signaling scaffold molecule and regulator of Gα protein signaling Rgs14a in coordinating the onset of migration with the presence of migration guidance cues. Furthermore, we show that this control level involves the regulation of the cell–cell adhesion molecule E-cadherin, a molecule implicated in motility acquisition in a range of normal physiological events and in disease conditions.

Summary Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent [1–3]. Intracellular pH gradients were shown to occur in certain cells migrating in vitro [4–6], but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types [7–9], including metastatic cancer cells [6], is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells.

The Hedgehog (Hh) pathway plays dual roles in proliferation and patterning during embryonic development, but the mechanism(s) that distinguish the mitogenic and patterning activities of Hh signalling are not fully understood. An additional level of complexity is provided by the observation that Hh signalling can both promote and inhibit cell proliferation. One model to account for this apparent paradox is that Hh signalling primarily regulates cell cycle kinetics, such that activation of Hh signalling promotes fast cycling and an earlier cell cycle exit. Here we report that activation of Hh signalling promotes endodermal cell proliferation but inhibits proliferation in neighbouring non-endodermal cells, suggesting that the cell cycle kinetics model is insufficient to account for the opposing proliferative responses to Hh signalling. We show that expression of the chemokine receptor Cxcr4a is a critical parameter that determines the proliferative response to Hh signalling, and that loss of Cxcr4a function attenuates the transcription of cell cycle regulator targets of Hh signalling without affecting general transcriptional targets. We show that Cxcr4a inhibits PKA activity independently of Hh signalling, and propose that Cxcr4a enhances Hh-dependent proliferation by promoting the activity of Gli1. Our results indicate that Cxcr4a is required for Hh-dependent cell proliferation but not for Hh-dependent patterning, and suggest that the parallel activation of Cxcr4a is required to modulate the Hh pathway to distinguish between patterning and proliferation.

Item does not contain fulltext A crucial regulator of Cxcl12 is the decoy receptor Cxcr7, which controls the level of the chemokine in the tissue. The molecular mechanisms that enable Cxcr7 to function as an efficient molecular sink are not known. Using zebrafish primordial germ cells as a model, we identify a novel role for beta-arrestins in controlling the intracellular trafficking of Cxcr7. beta-arrestins facilitate the recycling of Cxcr7 from late endosomal compartments back to the plasma membrane, whereas the internalized ligand undergoes lysosomal degradation. beta-arrestins thus function in regulating chemokine gradient formation, allowing responding cells to discriminate between alternative migration targets in vivo. 01 augustus 2012

SUMMARY Single-cell migration is a key process in development, homeostasis, and disease. Nevertheless, the control over basic cellular mechanisms directing cells into motile behavior in vivo is largely unknown. Here, we report on the identification of a minimal set of parameters the regulation of which confers proper morphology and cell motility. Zebrafish primordial germ cells rendered immotile by knockdown of Dead end, a negative regulator of miRNA function, were used as a platform for identifying processes restoring motility. We have defined myosin contractility, cell adhesion, and cortex properties as factors whose proper regulation is sufficient for restoring cell migration of this cell type. Tight control over the level of these cellular features, achieved through a balance between miRNA-430 function and the action of the RNA-binding protein Dead end, effectively transforms immotile primordial germ cells into polarized cells that actively migrate relative to cells in their environment.

Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus , germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE -LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE -LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE -LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.

In many species, primordial germ cells (PGCs) migrate from the position where they are specified to the site where the gonad develops, where they differentiate into sperm and egg. Germ cells thus serve as an excellent model for studying cell migration in the context of the live organism. In recent years, a number of cues directing the migration of the cells towards their target were identified and some of the relevant molecules and biochemical pathways were revealed. In this review we present those results, focusing on 'cell mechanics' of the process including cell adhesion, traction generation and cell polarization.

Primordial germ cell (PGC) development in Xenopus embryos relies on localised maternal determinants. We report on the identification and functional characterisation of such one novel activity, a germ plasm associated mRNA encoding for the Xenopus version of a kinesin termed KIF13B. Modulations of xKIF13B function result in germ cell mismigration and in reduced numbers of such cells. PGCs explanted from Xenopus embryos form bleb-like protrusions enriched in PIP3. Knockdown of xKIF13B results in inhibition of blebbing and PIP3 accumulation. Interference with PIP3 synthesis leads to PGC mismigration in vivo and in vitro. We propose that xKIF13B function is linked to polarized accumulation of PIP3 and directional migration of the PGCs in Xenopus embryos.

The Xenopus germ line is derived from a specialized region in the vegetal hemisphere of the oocyte, the germ plasm. Several maternal transcripts harboured in this region have been connected to the process of germ cell specification. We identified and functionally characterized a novel vegetally localizing mRNA encoding a glutamate receptor interacting protein (GRIP) family member in Xenopus, termed XGRIP2.1. XGRIP2.1 is specifically associated with the germ plasm and PGCs throughout Xenopus embryogenesis. Morpholino-mediated knockdown and overexpression of a putative dominant negative XGRIP2.1 protein fragment reduced average PGC numbers and interfered with the proper anteroposterior positioning of PGCs at tailbud stages. Thus, our results suggest that XGRIP2.1 is required for normal PGC development and migration in Xenopus.