Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system

Wnt5 signaling in vertebrate pancreas development

BACKGROUND: Signaling by the Wnt family of secreted glycoproteins through their receptors, the frizzled (Fz) family of seven-pass transmembrane proteins, is critical for numerous cell fate and tissue polarity decisions during development. RESULTS: We report a novel role of Wnt signaling in organogenesis using the formation of the islet during pancreatic development as a model tissue. We used the advantages of the zebrafish to visualize and document this process in living embryos and demonstrated that insulin-positive cells actively migrate to form an islet. We used morpholinos (MOs), sequence-specific translational inhibitors, and time-lapse imaging analysis to show that the Wnt-5 ligand and the Fz-2 receptor are required for proper insulin-cell migration in zebrafish. Histological analyses of islets in Wnt5a(-/- )mouse embryos showed that Wnt5a signaling is also critical for murine pancreatic insulin-cell migration. CONCLUSION: Our results implicate a conserved role of a Wnt5/Fz2 signaling pathway in islet formation during pancreatic development. This study opens the door for further investigation into a role of Wnt signaling in vertebrate organ development and disease

Lens Regeneration in Axolotl: New Evidence of Developmental Plasticity

Background: Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens

Ets1-Related Protein Is a Key Regulator of Vasculogenesis in Zebrafish

During embryonic development, multiple signaling pathways control specification, migration, and differentiation of the vascular endothelial cell precursors, angioblasts. No single gene responsible for the commitment of mesenchymal cells to the angioblast cell fate has been identified as yet. Here we report characterization and functional studies of Etsrp, a novel zebrafish ETS domain protein. etsrp embryonic expression is only restricted to vascular endothelial cells and their earliest precursors. Morpholino knockdown of Etsrp protein function resulted in the complete absence of circulation in zebrafish embryos. Angioblasts in etsrp–morpholino-injected embryos (morphants) failed to undergo migration and differentiation and did not coalesce into functional blood vessels. Expression of all vascular endothelial molecular markers tested was severely reduced in etsrp morphants, whereas hematopoietic markers were not affected. Overexpression of etsrp RNA caused multiple cell types to express vascular endothelial markers. etsrp RNA restored expression of vascular markers in cloche mutants, defective in hematopoietic and endothelial cell formation, arguing that etsrp functions downstream of cloche in angioblast formation. etsrp gene function was also required for endothelial marker induction by the vascular endothelial growth factor (vegf) and stem cell leukemia (scl/tal1). These results demonstrate that Etsrp is necessary and sufficient for the initiation of vasculogenesis

The EYA Tyrosine Phosphatase Activity Is Pro-Angiogenic and Is Inhibited by Benzbromarone

Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies

ETS Transcription Factors in Embryonic Vascular Development

At least thirteen ETS-domain transcription factors are expressed during embryonic hematopoietic or vascular development and potentially function in the formation and maintenance of the embryonic vasculature or blood lineages. This review summarizes our current understanding of the specific roles played by ETS factors in vasculogenesis and angiogenesis and the implications of functional redundancies between them

ETS Transcription Factors in Embryonic Vascular Development

At least thirteen ETS-domain transcription factors are expressed during embryonic hematopoietic or vascular development and potentially function in the formation and maintenance of the embryonic vasculature or blood lineages. This review summarizes our current understanding of the specific roles played by ETS factors in vasculogenesis and angiogenesis and the implications of functional redundancies between them

Molecular Analysis of Early Vasculogenesis in <i>etsrp</i> Morphants

<p>(A, B, E, F, I, K) Uninjected control embryo; (C, D, G, H, J, L) 8–10 ng <i>etsrp</i> MO2-injected embryo. Anterior is to the left in all panels. (A–H) Embryos were flat mounted with their yolk removed. (A–D) <i>scl</i> expression; six-somite (A,C) and ten-somite (B,D) stages. Note that the anterior domain of <i>scl</i> expression (arrows) is reduced and the trunk domain (arrowheads) is missing in <i>etsrp</i> morphants. (E–H) <i>fli1</i> expression; six-somite (E,G) and ten-somite (F,H) stages. Note that the anterior domain of <i>fli1</i> expression (arrows) is missing in the <i>etsrp</i> morphants, while the posterior domain is not affected. Also note that the trunk domain of <i>fli1</i> expression (arrowheads, F,H) is missing at the ten-somite stage in <i>etsrp</i> morphants. (I–L) Etsrp knockdown blocks angioblast migration towards the midline as assayed by <i>etsrp</i> expression at the 16-somite (I,J) and 20-somite (K,L) stages. (I,K) Uninjected control embryo; (J,L) 7.5 ng <i>etsrp</i> MO2-injected embryo. Dorsal view, anterior is to the left. Note that the midline stripe of angioblasts (arrows) is missing in <i>etsrp</i> morphants. Also notice more intense <i>etsrp</i> expression in pre-migratory angioblasts (arrowheads) in <i>etsrp</i> morphants as compared to control embryos.</p

etsrp Is Required for <i>vegf</i> and <i>scl</i> Signaling

<p>(A–D) Etsrp is required for Vegf signaling as assayed for <i>flk1</i> expression at 26 hpf. (A) Control uninjected embryo, (B) <i>vegf</i> RNA-injected embryo, (C) 7.5 ng of <i>etsrp</i> MO2-injected embryo, (D) <i>vegf</i> RNA- and <i>etsrp</i> MO2-co-injected embryo. Note that <i>vegf</i> RNA induces strong <i>flk1</i> expression in (B) while <i>vegf</i> RNA and <i>etsrp</i> MO co-injection results in loss of <i>flk1</i> expression in (D), similar to the <i>etsrp</i> morphant phenotype in (C). (E,F) Etsrp expression analysis in Vegf morphants at 26 hpf. (E) Control uninjected embryo; (F) 10.5 ng of <i>vegf</i> MO-injected embryo. Note that <i>vegf</i> morphants have lost <i>etsrp</i> expression in the intersegmental vessels (arrowhead, E). (G–J) Scl knockdown affects <i>gata1</i> but not <i>etsrp</i> expression at the 15-somite stage. Dorsal view, anterior is to the left. (G,I) Control uninjected embryo; (H,J) 10 ng <i>scl</i> UTR-MO-injected embryo. (G,H) <i>gata1</i> expression; (I,J) <i>etsrp</i> expression. (K–N) Etsrp is required for <i>scl</i> signaling in <i>clo</i> mutants as analyzed for <i>flk1</i> expression at the 15-somite stage. (K) Control uninjected embryo; (L) 7.5 ng <i>etsrp</i> MO2-injected embryo; (M) <i>scl</i> RNA-injected embryo; (N) <i>scl</i> RNA- and <i>etsrp</i> MO2-co-injected embryo. Note that <i>scl</i> RNA causes ectopic <i>flk1</i> expression in (M) which is lost upon knockdown of Etsrp in (N).</p