Anne Karine Lagendijk

Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development.

Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.

Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model.

BackgroundAberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour.MethodsWe fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish.ResultsIn the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature.ConclusionOur results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.

Macrophage development from HSCs requires PU.1-coordinated microRNA expression

The differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.

MicroRNA-23 Restricts Cardiac Valve Formation by Inhibiting Has2 and Extracellular Hyaluronic Acid Production

Rationale: Since their discovery almost 20 years ago, microRNAs have been shown to perform essential roles during tissue development and disease. Although roles for microRNAs in the myocardium during embryo development and cardiac disease have been demonstrated, very little is know about their role in the endocardium or during cardiac valve formation. Objective: To study the role of microRNAs in cardiac valve formation. Methods and Results: We show that zebrafish dicer mutant embryos, lacking mature miRNAs, form excessive endocardial cushions. By screening miRNAs expressed in the heart, we found that miR-23 is both necessary and sufficient for restricting the number of endocardial cells that differentiate into endocardial cushion cells. In addition, in mouse endothelial cells, miR-23 inhibited a transforming growth factor-&bgr;–induced endothelial-to-mesenchymal transition. By in silico screening of expression data with predicted miR-23 target sites combined with in vivo testing, we identified hyaluronic acid synthase 2 (Has2), Icat, and Tmem2 as novel direct targets of miR-23. Finally, we demonstrate that the upregulation of Has2, an extracellular remodeling enzyme required for endocardial cushion and valve formation, is responsible for the excessive endocardial cushion cell differentiation in dicer mutants. Conclusions: MiR-23 in the embryonic heart is required to restrict endocardial cushion formation by inhibiting Has2 expression and extracellular hyaluronic acid production.

Bmp Signaling Exerts Opposite Effects on Cardiac Differentiation

Rationale: The importance for Bmp signaling during embryonic stem cell differentiation into myocardial cells has been recognized. The question when and where Bmp signaling in vivo regulates myocardial differentiation has remained largely unanswered. Objective: To identify when and where Bmp signaling regulates cardiogenic differentiation. Methods and Results: Here we have observed that in zebrafish embryos, Bmp signaling is active in cardiac progenitor cells prior to their differentiation into cardiomyocytes. Bmp signaling is continuously required during somitogenesis within the anterior lateral plate mesoderm to induce myocardial differentiation. Surprisingly, Bmp signaling is actively repressed in differentiating myocardial cells. We identified the inhibitory Smad6a, which is expressed in the cardiac tissue, to be required to inhibit Bmp signaling and thereby promote expansion of the ventricular myocardium. Conclusion: Bmp signaling exerts opposing effects on myocardial differentiation in the embryo by promoting as well as inhibiting cardiac growth.

A Nodal-independent and tissue-intrinsic mechanism controls heart-looping chirality

Breaking left-right symmetry in bilateria is a major event during embryo development that is required for asymmetric organ position, directional organ looping and lateralized organ function in the adult. Asymmetric expression of Nodal-related genes is hypothesized to be the driving force behind regulation of organ laterality. Here we identify a Nodal-independent mechanism that drives asymmetric heart looping in zebrafish embryos. In a unique mutant defective for the Nodal-related southpaw gene, preferential dextral looping in the heart is maintained, whereas gut and brain asymmetries are randomized. As genetic and pharmacological inhibition of Nodal signalling does not abolish heart asymmetry, a yet undiscovered mechanism controls heart chirality. This mechanism is tissue intrinsic, as explanted hearts maintain ex vivo retain chiral looping behaviour and require actin polymerization and myosin II activity. We find that Nodal signalling regulates actin gene expression, supporting a model in which Nodal signalling amplifies this tissue-intrinsic mechanism of heart looping.

Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.

Transmembrane protein 2 (Tmem2) is required to regionally restrict atrioventricular canal boundary and endocardial cushion development

The atrioventricular canal (AVC) physically separates the atrial and ventricular chambers of the heart and plays a crucial role in the development of the valves and septa. Defects in AVC development result in aberrant heart morphogenesis and are a significant cause of congenital heart malformations. We have used a forward genetic screen in zebrafish to identify novel regulators of cardiac morphogenesis. We isolated a mutant, named wickham (wkm), that was indistinguishable from siblings at the linear heart tube stage but exhibited a specific loss of cardiac looping at later developmental stages. Positional cloning revealed that the wkm locus encodes transmembrane protein 2 (Tmem2), a single-pass transmembrane protein of previously unknown function. Expression analysis demonstrated myocardial and endocardial expression of tmem2 in zebrafish and conserved expression in the endocardium of mouse embryos. Detailed phenotypic analysis of the wkm mutant identified an expansion of expression of known myocardial and endocardial AVC markers, including bmp4 and has2. By contrast, a reduction in the expression of spp1, a marker of the maturing valvular primordia, was observed, suggesting that an expansion of immature AVC is detrimental to later valve maturation. Finally, we show that immature AVC expansion in wkm mutants is rescued by depleting Bmp4, indicating that Tmem2 restricts bmp4 expression to delimit the AVC primordium during cardiac development.

mafba is a downstream transcriptional effector of Vegfc signaling essential for embryonic lymphangiogenesis in zebrafish.

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.

Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues

Summary Non-coding microRNA (miRNA) molecules bind their target mRNAs and thereby modulate the amount of protein produced. To understand the significance of a potential miRNA-mRNA interaction, temporal and spatial information on miRNA and mRNA expression is essential. Here, we provide a detailed protocol for miRNA whole mount in situ hybridization. We introduce the use of Morpholino based oligos as antisense probes for miRNA detection, in addition to the current “gold standard” locked nucleic acid (LNA) probes. Furthermore we have modified existing miRNA in situ protocols thereby improving both sensitivity and resolution of miRNA visualization in whole zebrafish embryos and adult tissues.