Lymphatic vessels arise from specialized angioblasts within a venous niche.

How cells acquire their fate is a fundamental question in developmental and regenerative biology. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein are thought to generate lymphatic vessels through trans-differentiation. Here we show that in zebrafish, lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the cardinal vein, which also generates arterial and venous fates. We further identify Wnt5b as a novel lymphatic inductive signal and show that it also promotes the 'angioblast-to-lymphatic' transition in human embryonic stem cells, suggesting that this process is evolutionarily conserved. Our results uncover a novel mechanism of lymphatic specification, and provide the first characterization of the lymphatic inductive niche. More broadly, our findings highlight the cardinal vein as a heterogeneous structure, analogous to the haematopoietic niche in the aortic floor.

Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish.

Recent evidence indicates a specific role for vascular endothelial growth factor a (Vegfa) during artery development in both zebrafish and mouse embryos, whereas less is known about signals that govern vein formation. In zebrafish, loss of vegfa blocks segmental artery formation and reduces artery-specific gene expression, whereas veins are largely unaffected. Here, we describe a mutation in the zebrafish vegf receptor-2 homolog, kdra, which eliminates its kinase activity and leads to specific defects in artery development. We further find that Flt4, a receptor for Vegfc, cooperates with Kdr during artery morphogenesis, but not differentiation. We also identify an additional zebrafish vegfr-2 ortholog, referred to as kdrb, which can partially compensate for loss of kdra but is dispensable for vascular development in wild-type embryos. Interestingly, we find that these Vegf receptors are also required for formation of veins but in distinct genetic interactions that differ from those required for artery development. Taken together, our results indicate that formation of arteries and veins in the embryo is governed in part by different Vegf receptor combinations and suggest a genetic mechanism for generating blood vessel diversity during vertebrate development.

Notch signaling is required for arterial-venous differentiation during embryonic vascular development.

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. We show that loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, we find that ectopic activation of Notch signaling leads to repression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. Our results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries. Movies available on-line

Modulation of a calcium/calmodulin-dependent protein kinase cascade by retinoic acid during neutrophil maturation.

Retinoic acid is a lipophilic derivative of vitamin A that can cause differentiation in a variety of cell types. A large body of evidence has shown that normal retinoid signaling is required for proper neutrophil maturation in vitro and in vivo. In this study, we have found that calcium/calmodulin dependent (CaM) protein kinase kinase alpha (CaMKKalpha) is upregulated in an immediate early fashion during retinoic acid induced neutrophil maturation. Furthermore, we describe the expression and modulation of various components of the CaM kinase cascade during neutrophil maturation. We have confirmed upregulation of CaMKKalpha protein by Western analysis and further show that CaMKKbeta is expressed, although its protein levels are constant throughout induction. We also find that neutrophil progenitor cells express both CaMKI and CaMKIV transcripts. RNase protection and Western analysis show that CaMKIV is downregulated during neutrophil maturation. In contrast, CaMKI transcript and protein is expressed in uninduced cells and is induced by all-trans retinoic acid. These data represent the first report of a CaM kinase cascade in myeloid cells and suggests that this cascade may mediate some of the well-characterized effects of calcium on neutrophil function. These observations also support the idea that the retinoic acid receptors play a major role in mediating neutrophil specific gene expression and differentiation.

Neutrophil maturation and the role of retinoic acid.

Neutrophil maturation occurs in well defined morphological stages that correlate with the acquisition of molecular markers associated with neutrophil function. A variety of factors are known to play a role in terminal neutrophil maturation, including the vitamin A derivative, retinoic acid. Retinoic acid can directly modulate gene expression via binding to its nuclear receptors, which can, in turn, activate transcription of target genes. A role for retinoic acid during neutrophil maturation has been suggested from a variety of sources. Here we present a review of the mechanism of retinoic acid receptor action and the major evidence showing that normal retinoid signaling is required for neutrophil maturation.

Normal neutrophil differentiation and secondary granule gene expression in the EML and MPRO cell lines.

The EML and MPRO cell lines express a dominant negative retinoic acid receptor alpha that causes a block at specific stages of myelopoiesis. The EML cell line is multipotent and gives rise to erythroid, lymphoid, and myeloid lineages depending on the presence of appropriate cytokines. The MPRO cell line is promyelocytic and undergoes neutrophilic differentiation when induced with all-trans retinoic acid in the presence of granulocyte/macrophage colony-stimulating factor. Previous studies have shown that both of these cell lines undergo morphological differentiation into neutrophils. In this study, we show that unlike other models of neutrophil differentiation such as NB4 and HL60, both EML and MPRO cell lines undergo complete, normal granulocytic differentiation programs. Similar to HL60, MPRO and EML induce expression of CD11b/CD18 and also exhibit downregulation of CD34 on differentiation. In contrast to HL60 and NB4, EML and MPRO cell lines coordinately upregulate secondary granule transcripts for lactoferrin and neutrophil gelatinase. Furthermore, we have confirmed previous observations that serum can induce a low level of differentiation in MPRO cells and that it is possible to grow these cells in serum-free medium, thereby eliminating this effect. Based on these studies, it appears that these lines can serve as a model for normal retinoic acid-induced neutrophil differentiation and provide insight into the role of the retinoic acid-responsive pathway in normal and leukemic myelopoiesis.

Representational difference analysis of a committed myeloid progenitor cell line reveals evidence for bilineage potential.

In this study we have sought to characterize a committed myeloid progenitor cell line in an attempt to isolate general factors that may promote differentiation. We used cDNA representational difference analysis (RDA), which allows analysis of differential gene expression, to compare EML and EPRO cells. We have isolated nine differentially expressed cDNA fragments as confirmed by slot blot, Northern, and PCR analysis. Three of nine sequences appear to be novel whereas the identity of the remaining fragments suggested that the EPRO cell line is multipotent. Among the isolated sequences were eosinophilic, monocytic, and neutrophilic specific genes. Therefore, we tested the ability of EPRO cells to differentiate along multiple myeloid lineages and found that EPRO cells exhibited morphologic maturation into either monocyte/macrophages or neutrophils, but not eosinophils. Furthermore, when EPRO cells were exposed to ATRA, neutrophil specific genes were induced, whereas monocytic markers were induced by phorbol ester treatment. This study highlights the use of cDNA RDA in conjunction with the EML/EPRO cell line to isolate markers associated with macrophage and neutrophil differentiation and establishes the usefulness of this system in the search for factors involved in myeloid commitment.

Isolation and characterization of the cDNA for mouse neutrophil collagenase: demonstration of shared negative regulatory pathways for neutrophil secondary granule protein gene expression.

A characteristic of normal neutrophil maturation is the induction of secondary granule protein (SGP) mRNA expression. Several leukemic human cell lines mimic normal morphologic neutrophil differentiation but fail to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase (NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to differentiate into neutrophils and induce expression of LF and NG. Therefore, to study the normal regulation and function of these genes, the corresponding murine homologs must be isolated. Using cDNA representational difference analysis (RDA) to compare a committed myeloid progenitor cell line (EPRO) with the multipotent stem cell line from which it was derived (EML), we isolated a fragment bearing homology to human neutrophil collagenase (hNC). Here, we describe the cloning and characterization of a full-length ( approximately 2 kb) clone that exhibits nearly 65% nucleotide and 73% amino acid identity to hNC. Ribonuclease protection analysis (RPA) of the tissues and cell lines shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the mouse homolog of hNC. Furthermore, we have demonstrated a shared negative regulatory pathway for this and other SGP genes. We have previously shown that CCAAT displacement protein (CDP/cut) binds to a specific region of the LF promoter, and overexpression of CDP blocks G-CSF-induced upregulation of LF gene expression in 32D C13 cells. We show here that in these cells, upregulation of both NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.