Zebrafish microRNA-126 determines hematopoietic cell fate through c-Myb.

Precise regulatory mechanisms are required to appropriately modulate the cellular levels of transcription factors controlling cell fate decisions during blood cell development. In this study, we show that miR-126 is a novel physiological regulator of the proto-oncogene c-myb during definitive hematopoiesis. We show that knockdown of miR-126 results in increased c-Myb levels and promotes erythropoiesis at the expense of thrombopoiesis in vivo. We further provide evidence that specification of thrombocyte versus erythrocyte cell lineages is altered by the concerted activities of the microRNAs (miRNAs) miR-126 and miR-150. Both miRNAs are required but not sufficient individually to precisely regulate the cell fate decision between erythroid and megakaryocytic lineages during definitive hematopoiesis in vivo. These results support the notion that miRNAs not only function to provide precision to developmental programs but also are essential determinants in the control of variable potential functions of a single gene during hematopoiesis.

NOTCH1-induced T-cell leukemia in transgenic zebrafish.

Activating mutations in the NOTCH1 gene have been found in about 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL). In order to study the molecular mechanisms by which altered Notch signaling induces leukemia, a zebrafish model of human NOTCH1-induced T-cell leukemia was generated. Seven of sixteen mosaic fish developed a T-cell lymphoproliferative disease at about 5 months. These neoplastic cells extensively invaded tissues throughout the fish and caused an aggressive and lethal leukemia when transplanted into irradiated recipient fish. However, stable transgenic fish exhibited a longer latency for leukemia onset. When the stable transgenic line was crossed with another line overexpressing the zebrafish bcl2 gene, the leukemia onset was dramatically accelerated, indicating synergy between the Notch pathway and the bcl2-mediated antiapoptotic pathway. Reverse transcription-polymerase chain reaction analysis showed that Notch target genes such as her6 and her9 were highly expressed in NOTCH1-induced leukemias. The ability of this model to detect a strong interaction between NOTCH1 and bcl2 suggests that genetic modifier screens have a high likelihood of revealing other genes that can cooperate with NOTCH1 to induce T-ALL.

Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio).

Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia.

Zebrafish myelopoiesis and blood cell development.

The zebrafish (Danio rerio) animal model offers a unique opportunity to discover novel genes required for the control of normal vertebrate myeloid cell development. It is well suited for both developmental and genetic analyses: eg, genome-wide chemical mutagenesis screens have led to the identification of specific new genes affecting vertebrate erythropoiesis. Mutants defective in one or more hematopoietic functions will be useful as models of human disease and will assist in the elucidation of lineage-specific developmental programs. By using a combination of forward genetic mutagenesis screens and emerging strategies based on transgenic and antisense knockdown approaches, it should be possible to dissect the genetic programs that lead to myeloproliferative/myelodysplastic syndromes and to acute myeloid leukemia.

Myelopoiesis in the zebrafish, Danio rerio.

Genome-wide chemical mutagenesis screens in the zebrafish (Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafish mpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, and L-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis. (Blood. 2001;98:643-651)

Growth cones utilize both widespread and local directional cues in the zebrafish brain.

The distribution of cues that provide directional information for specific growth cones in the zebrafish brain was functionally assayed by transplanting epiphysial neurons to ectopic locations in the embryonic brain followed by determining the pathways taken by the donor axons. Epiphysial axons normally first extend ventrally from their position in the dorsal diencephalon and then turn and extend anteriorly in the ventral diencephalon. When transplanted to ectopic sites at other axial levels of the brain, where in principle the axons could extend in any direction, epiphysial axons consistently extended ventrally. Furthermore, following initial ventral extension ectopic epiphysial axons turned randomly in the anterior and posterior directions. These results suggest that the cues for ventral extension are widely distributed along the rostrocaudal axis of the zebrafish brain, but the cues for subsequent anterior extension are restricted to the site where the epiphysial axons normally turn longitudinally.

Molecular cloning and developmental expression of a zebrafish axonal glycoprotein similar to TAG-1.

TAG-1 is a mammalian cell adhesion molecule of the immunoglobulin superfamily that is expressed transiently by a subset of neurons and serves as a fertile substrate for neurite outgrowth in vitro (Furley, A.H., Morton, S.B., Manalo, D., Karagogeos, S., Dodd, H., Jessell, T.M., 1990 The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth promoting activity. Cell 61, 157-170). In order to examine the in vivo function of this molecule, we have cloned a zebrafish tag1-like cDNA and analyzed its expression patterns. tag1 Is expressed transiently by specific subsets of neurons when they are projecting their axons or when they are migrating. The specific and dynamic pattern of expression of zebrafish tag1 is consistent with its proposed role in axon guidance and cell migration.

The development of the posterior body in zebrafish.

In order to understand the developmental mechanisms of posterior body formation in the zebrafish, a fate map of the zebrafish tailbud was generated along with a detailed analysis of tailbud cell movements. The fate map of the zebrafish tailbud shows that it contains tissue-restricted domains and is not a homogeneous blastema. Furthermore, time-lapse analysis shows that some cell movements and behaviors in the tailbud are similar to those seen during gastrulation, while others are unique to the posterior body. The extension of axial mesoderm and the continuation of ingression throughout zebrafish tail development suggests the continuation of processes initiated during gastrulation. Unique properties of zebrafish posterior body development include the bilateral distribution of tailbud cell progeny and the exhibition of different forms of ingression within specific tailbud domains. The ingression of cells in the anterior tailbud only gives rise to paraxial mesoderm, at the exclusion of axial mesoderm. Cells of the posterior tailbud undergo subduction, a novel form of ingression resulting in the restriction of this tailbud domain to paraxial mesodermal fates. The intermixing of spinal cord and muscle precursor cells, as well as evidence for pluripotent cells within the tailbud, suggest that complex inductive mechanisms accompany these cell movements throughout tail elongation. Rates of cell proliferation in the tailbud were examined and found to be relatively low at the tip of the tail indicating that tail elongation is not due to growth at its posterior end. However, higher rates of cell proliferation in the dorsomedial region of the tail may contribute to the preferential posterior movement of cells in this tailbud region and to the general extension of the tail. Understanding the cellular movements, cell fates and gene expression patterns in the tailbud will help to determine the nature of this important aspect of vertebrate development.

The molecular cloning and characterization of potential chick DM-GRASP homologs in zebrafish and mouse.

A full-length zebrafish cDNA clone and a partial mouse cDNA clone similar to chick DM-GRASP were isolated and analyzed. The nucleotide sequence of the full-length zebrafish clone shares 54% identity, and predicts 39% amino acid identity, with chick DM-GRASP. The partial mouse clone shares 76% nucleotide identity, and predicts 76% amino acid identity, with chick DM-GRASP. The predicted proteins encoded by both of these clones exhibit conserved structural domains that are characteristic of the chick protein. These features may identify them as a distinct subfamily within the immunoglobulin superfamily of cell adhesion molecules. Expression of the zebrafish DM-GRASP protein is similar to chick DM-GRASP and is principally restricted to a small subset of developing sensory and motor neurons during axonogenesis. Zebrafish DM-GRASP expression was temporally regulated and limited to specific axon domains. This regional expression correlated with fasciculated axon domains. These results suggest that the zebrafish and mouse cDNA clones represent the respective fish and mammalian homologs of chick DM-GRASP. The highly selective expression of zebrafish DM-GRASP suggests that it is involved in the selective fasciculation and guidance of axons along their normal pathways.

The cell cycle dependence of the secretory pathway in developing Xenopus laevis.

The cell cycle during the cleavage period of the amphibian Xenopus laevis is about 30 min long and oscillates between equal periods of mitosis and interphase. At the midblastula transition (MBT) the length of interphase begins to elongate and brings about corresponding changes in the activities of cell cycle-dependent processes. In this study protein secretion and Golgi processing during embryonic Xenopus development were examined. The elongation of interphase, either during normal development or experimentally induced, resulted in an increase in the secretion of both endogenous and exogenous proteins. Secretion was found to increase linearly with the increase in interphase length, indicating that the rate of secretion was constant and was regulated by the length of interphase. M-phase arrest in embryos and oocytes produced an inhibition of protein secretion that was reversible if the cell cycle was returned to interphase. This M-phase block of the secretory pathway was found to take place between the trans Golgi compartment and the plasma membrane. The developmental increase in the function of this pathway after the MBT may affect the expression of surface and secreted proteins important for the cell-cell interactions necessary for subsequent development through gastrulation.

The cell cycle dependence of protein synthesis during Xenopus laevis development.

The regulation of early embryonic development in the amphibian Xenopus laevis depends largely upon translational and post-translational regulatory mechanisms to direct the complex cytodifferentiations that take place during early cleavage and blastula formation. The cell cycle dependence of protein synthesis was examined in developing Xenopus embryos as well as in cycling cell-free lysates from Xenopus eggs. In both cases M-phase and the activation of the M-phase kinase were found to be correlated with an inhibition of translation. Translation in both the rough endoplasmic reticulum and cytosolic-free ribosomes were affected by this inhibition. Since elongation was found to be unaffected by M-phase, shifts in the polysome profiles during M-phase indicated that the inhibition affected initiation processes. The activity of the M-phase kinase may inhibit initiation through the modification of initiation factors or some other component during this process. The cell cycle dependence of translation may affect developmental mechanisms controlled by the titration of regulatory proteins.

Progression from meiosis I to meiosis II in Xenopus oocytes requires de novo translation of the mosxe protooncogene.

The meiotic maturation of Xenopus oocytes exhibits an early requirement for expression of the mosxe protooncogene. The mosxe protein has also been shown to be a component of cytostatic factor (CSF), which is responsible for arrest at metaphase of meiosis II. In this study, we have assayed the appearance of CSF activity in oocytes induced to mature either by progesterone treatment or by overexpression of mosxe. Progesterone-stimulated oocytes did not exhibit CSF activity until 30-60 min after germinal vesicle breakdown (GVBD). Both the appearance of CSF activity and the progression from meiosis I to meiosis II were inhibited by microinjection of mosxe antisense oligonucleotides just prior to GVBD. These results demonstrate a translational requirement for mosxe, which is temporally distinct from the requirement for mosxe expression at the onset of meiotic maturation. In contrast to progesterone-treated oocytes, oocytes that were induced to mature by overexpression of mosxe exhibited CSF activity at least 3 hr prior to GVBD. Despite the early appearance of CSF, these oocytes were not arrested at meiosis I. These results indicate that, although CSF activity is capable of stabilizing maturation-promoting factor (MPF) at meiosis II and in cleaving embryos, it is incapable of stabilizing MPF prior to or at meiosis I. These studies show that the complex regulation of the cell cycle during meiosis differs significantly from the regulation of the cell cycle during mitosis.

Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos.

Previous work has demonstrated that the Xenopus protooncogene mosxe can induce the maturation of prophase-arrested Xenopus oocytes. Recently, we showed that mosxe can transform murine NIH3T3 fibroblasts, although it exhibited only 1-2% of the transforming activity of the v-mos oncogene. In this study we have investigated the ability of the v-mos protein to substitute for the mosxe protein in stimulating Xenopus oocytes to complete meiosis. Microinjection of in vitro synthesized RNAs encoding either the mosxe or v-mos proteins stimulates resting oocytes to undergo germinal vesicle breakdown. Microinjection of an antisense oligonucleotide spanning the initiation codon of the mosxe gene blocked progesterone-induced oocyte maturation. When oocytes were microinjected first with the mosxe antisense oligonucleotide, and subsequently with in vitro synthesized v-mos RNA, meiotic maturation was rescued as evidenced by germinal vesicle breakdown. The v-mos protein exhibited in vitro kinase activity when recovered by immunoprecipitation from either microinjected Xenopus oocytes or transfected monkey COS-1 cells; however, in parallel experiments, we were unable to detect in vitro kinase activity associated with the mosxe protein. Microinjection of in vitro synthesized v-mos RNA into cleaving Xenopus embryos resulted in mitotic arrest, demonstrating that the v-mos protein can function like the mosxe protein as a component of cytostatic factor. These results exemplify the apparently conflicting effects of the v-mos protein, namely, its ability to induce maturation of oocytes, its ability to arrest mitotic cleavage of Xenopus embryo, and its ability to transform mammalian fibroblasts.

Xenopus homolog of the mos protooncogene transforms mammalian fibroblasts and induces maturation of Xenopus oocytes.

The oncogene v-mos transforms mammalian fibroblasts and encodes a serine/threonine protein kinase. Expression of the c-mos protooncogene is most abundant in germ cells, suggesting a normal role for c-mos in meiosis. Here we describe the isolation of cDNA clones containing the complete coding region of the Xenopus laevis homolog of c-mos (mosxe). The mosxe gene is transforming when introduced into murine NIH 3T3 cells, and transformation is abrogated by a lysine-to-arginine mutation in the canonical ATP-binding site. Microinjection of in vitro transcribed mosxe RNA into prophase-arrested Xenopus oocytes causes a resumption of meiosis, leading to germinal vesicle breakdown and oocyte maturation. Oocyte maturation was not observed after microinjection of in vitro transcribed mosxe RNA encoding the lysine-to-arginine mutation. These results demonstrate that the mosxe-encoded protein can induce progression through the cell cycle for both meiotic and mitotic cells and that this property is dependent on the presumptive ATP-binding domain in the protein kinase.