Transcription factor Meis1b regulates craniofacial morphogenesis in zebrafish.

BACKGROUND: Meis family of transcription factors operates in Pbx-Meis-Hox regulatory network controlling development of various tissues including eye, limbs, heart, hindbrain or craniofacial skeletal elements originating from the neural crest. Although studies in mouse provide abundant information about Meis factors function in embryogenesis, little is known about their role in zebrafish. RESULTS: We generated zebrafish lines carrying null mutations in meis1a, meis1b, meis2a, and meis2b genes. Only meis1b mutants are lethal at larval stage around 13 dpf whereas the other mutant lines are viable and fertile. We focused on development of neural crest-derived craniofacial structures such as tendons, cranial nerves, cartilage and accompanying muscles. Meis1b mutants displayed morphogenetic abnormalities in the cartilage originating from the first and second pharyngeal arches. Meckel's cartilage was shorter and wider with fused anterior symphysis and abnormal chondrocyte organization. This resulted in impaired tendons and muscle fiber connections while tenocyte development was not largely affected. CONCLUSIONS: Loss-of-function mutation in meis1b affects cartilage morphology in the lower jaw that leads to disrupted organization of muscles and tendons.

M-CSFR/CSF1R signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands.

Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. The CSF1R pathway is a promising therapeutic target in many human diseases, including neurological disorders and cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands (Csf1a, Csf1b, and Il34) in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors, Csf1ra and Csf1rb, however there is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Furthermore, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation, resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rbΔ4bp-deficient and il34Δ5bp-deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors, and the expression of csf1ra and csf1rb is nonoverlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.

Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion.

Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

Zebrabase: An Intuitive Tracking Solution for Aquatic Model Organisms.

Small fish species, such as zebrafish and medaka, are increasingly gaining popularity in basic research and disease modeling as a useful alternative to rodent model organisms. However, the tracking options for fish within a facility are rather limited. In this study, we present an aquatic species tracking database, Zebrabase, developed in our zebrafish research and breeding facility that represents a practical and scalable solution and an intuitive platform for scientists, fish managers, and caretakers, in both small and large facilities. Zebrabase is a scalable, cross-platform fish tracking database developed especially for fish research facilities. Nevertheless, this platform can be easily adapted for a wide variety of aquatic model organisms housed in tanks. It provides sophisticated tracking, reporting, and management functions that help keep animal-related records well organized, including a QR code functionality for tank labeling. The implementation of various user roles ensures a functional hierarchy and customized access to specific functions and data. In addition, Zebrabase makes it easy to personalize rooms and racks, and its advanced statistics and reporting options make it an excellent tool for creating periodic reports of animal usage and productivity. Communication between the facility and the researchers can be streamlined by the database functions. Finally, Zebrabase also features an interactive breeding history and a smart interface with advanced visualizations and intuitive color coding that accelerate the processes.

Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.

This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.

Meis2 is essential for cranial and cardiac neural crest development.

BACKGROUND: TALE-class homeodomain transcription factors Meis and Pbx play important roles in formation of the embryonic brain, eye, heart, cartilage or hematopoiesis. Loss-of-function studies of Pbx1, 2 and 3 and Meis1 documented specific functions in embryogenesis, however, functional studies of Meis2 in mouse are still missing. We have generated a conditional allele of Meis2 in mice and shown that systemic inactivation of the Meis2 gene results in lethality by the embryonic day 14 that is accompanied with hemorrhaging. RESULTS: We show that neural crest cells express Meis2 and Meis2-defficient embryos display defects in tissues that are derived from the neural crest, such as an abnormal heart outflow tract with the persistent truncus arteriosus and abnormal cranial nerves. The importance of Meis2 for neural crest cells is further confirmed by means of conditional inactivation of Meis2 using crest-specific AP2α-IRES-Cre mouse. Conditional mutants display perturbed development of the craniofacial skeleton with severe anomalies in cranial bones and cartilages, heart and cranial nerve abnormalities. CONCLUSIONS: Meis2-null mice are embryonic lethal. Our results reveal a critical role of Meis2 during cranial and cardiac neural crest cells development in mouse.

Prolyl Isomerase Pin1 Regulates Axon Guidance by Stabilizing CRMP2A Selectively in Distal Axons.

Axon guidance relies on precise translation of extracellular signal gradients into local changes in cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here, we show that during embryonic development in growing axons, a low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A levels specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo, leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance.

Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin.

In nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.

Monensin inhibits canonical Wnt signaling in human colorectal cancer cells and suppresses tumor growth in multiple intestinal neoplasia mice.

The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/ß-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with ß-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or ß-catenin-induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of ß-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling.

A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.

Increased nuclear accumulation of ß-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/ß-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the ß-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the ß-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the ß-catenin destruction complex, followed by increased degradation of ß-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of ß-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/ß-catenin signaling through inhibiting the PARP domain of TNKS1/2.

Mouse Tcf3 represses canonical Wnt signaling by either competing for ß-catenin binding or through occupation of DNA-binding sites.

Tcf3 acts as a transcription factor controlling gene expression in canonical Wnt signaling. In this study we show that mouse Tcf3 represses canonical Wnt signaling in mouse neural stem cells and in human HEK 293 cells. We demonstrate that mouse Tcf3 mediates repression of both moderate and high levels of canonical Wnt signaling, by either competing with other members of the Tcf/Lef family for binding to ß-catenin, or for binding to DNA. We observed that the repressor activity of mouse Tcf3 was only relieved effectively upon simultaneous disruption of both mechanisms. Immunofluorescence of transfected HEK 293 cells showed co-localization of ß-catenin and Tcf3 in the nucleus of cells transfected with full-length Tcf3, but not in cells transfected with N-terminal deleted versions. A direct physical interaction between ß-catenin and Tcf3 in the nucleus was confirmed by co-immunoprecipitation studies. The inhibitory ß-catenin/Tcf3 interface was independent of the ability of Tcf3 to directly interact with DNA.

Novel synthetic antagonists of canonical Wnt signaling inhibit colorectal cancer cell growth.

Canonical Wnt signaling is deregulated in several types of human cancer where it plays a central role in tumor cell growth and progression. Here we report the identification of 2 new small molecules that specifically inhibit canonical Wnt pathway at the level of the destruction complex. Specificity was verified in various cellular reporter systems, a Xenopus double-axis formation assay and a gene expression profile analysis. In human colorectal cancer (CRC) cells, the new compounds JW67 and JW74 rapidly reduced active ß-catenin with a subsequent downregulation of Wnt target genes, including AXIN2, SP5, and NKD1. Notably, AXIN2 protein levels were strongly increased after compound exposure. Long-term treatment with JW74 inhibited the growth of tumor cells in both a mouse xenograft model of CRC and in Apc(Min) mice (multiple intestinal neoplasia, Min). Our findings rationalize further preclinical and clinical evaluation of these new compounds as novel modalities for cancer treatment.

A dynamic gradient of Wnt signaling controls initiation of neurogenesis in the mammalian cortex and cellular specification in the hippocampus.

Neurogenesis in the developing neocortex is a strictly regulated process of cell division and differentiation. Here we report that a gradual retreat of canonical Wnt signaling in the cortex from lateral-to-medial and anterior-to-posterior is a prerequisite of neurogenesis. Ectopic expression of a beta-catenin/LEF1 fusion protein maintains active canonical Wnt signaling in the developing cortex and delays the expression onset of the neurogenic factors Pax6, Ngn2 and Tbr2 and subsequent neurogenesis. Contrary to this, conditional ablation of beta-catenin accelerates expression of the same neurogenic genes. Furthermore, we show that a sustained canonical Wnt activity in the lateral cortex gives rise to cells with hippocampal characteristics in the cortical plate at the expense of the cortical fate, and to cells with dentate gyrus characteristics in the hippocampus. This suggests that the dose of canonical Wnt signaling determines cellular fate in the developing cortex and hippocampus, and that recession of Wnt signaling acts as a morphogenetic gradient regulating neurogenesis in the cortex.