SUMMARYHere, we show that a novel Rspo1-Wnt-Vegfc-Vegfr3 signaling pathway plays an essential role in developmental angiogenesis. A mutation in R-spondin1 (rspo1), a Wnt signaling regulator, was uncovered during a forward-genetic screen for angiogenesisdeficient mutants in the zebrafish. Embryos lacking rspo1 or the proposed rspo1 receptor kremen form primary vessels by vasculogenesis, but are defective in subsequent angiogenesis. Endothelial cell-autonomous inhibition of canonical Wnt signaling also blocks angiogenesis in vivo. The pro-angiogenic effects of Rspo1/Wnt signaling are mediated by Vegfc/Vegfr3(Flt4) signaling. Vegfc expression is dependent on Rspo1 and Wnt, and Vegfc and Vegfr3 are necessary to promote angiogenesis downstream from Rspo1-Wnt. As all of these molecules are expressed by the endothelium during sprouting stages, these results suggest that Rspo1-Wnt-VegfC-Vegfr3 signaling plays a crucial role as an endothelial-autonomous permissive cue for developmental angiogenesis.

Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain.Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1 W84X/W84X embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval "bloodless" phase and develop to fertile adults with multilineage hematopoiesis. Using cd41-green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1 W84X/W84X embryos developed cd41 ؉ HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1. (Blood. 2010;115(14): 2806-2809) IntroductionRunx1 plays a critical role in the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) at the beginning of definitive hematopoiesis. 1 The function of Runx1 in adult HSCs, however, has not been fully resolved. Some studies suggest that Runx1 is required for the maintenance of HSCs, whereas others indicate that it negatively regulates the number of HSCs. 2,3 Overall, though, it has been difficult to explore the role of Runx1 in initiation of adult hematopoiesis because of limitations of conditional knockout mouse models. We recently reported on the generation of a zebrafish line, W84X, with a premature truncation in the runt domain of the runx1 protein. 4 This truncation removes most of the residues important in runx1 activity, such as CBF␤ and DNA binding, and nuclear localization signal. 5,6 Consistent with the Runx1 knockout mice, 7 the mutant embryos had normal primitive hematopoiesis but blockage of definitive hematopoiesis. 4 In this study, we analyzed the role of runx1 in adult hematopoiesis using the W84X mutant line. MethodsZebrafish were maintained under an approved National Institutes of Health animal use protocol. All zebrafish handling and breedings were performed as described. 8 Peripheral blood smears were prepared from tail clips of killed fish. Whole kidney marrow preparation and flow cytometric analysis were performed as described. 9 Killed adult fish were fixed in 10% formalin for histology. Lineage tracing by uncaging was performed as described. 10 Protocols for genotyping, microscopy and imaging, morpholino design, microinjections, reverse-transcription polymerase chain reaction (RT-PCR), and time lapse are described in the supplemental Methods (available on the Blood website; see the Supplemental Materials link at the top of the online article). Results and discussionAll runx1 W84X/W84X embryos lack definitive hematopoiesis, as demonstrated by lack of expression of HSC and lineage-specific markers between 36 hours after fertilization and 5 days after fertilization 4 (supplemental Figure 1A-G). Despite lack of runx1-expressing cells b...

Neural crest cells migrate throughout the embryo, but how cells move in a directed and collective manner has remained unclear. Here, we perform the first single-cell transcriptome analysis of cranial neural crest cell migration at three progressive stages in chick and identify and establish hierarchical relationships between cell position and time-specific transcriptional signatures. We determine a novel transcriptional signature of the most invasive neural crest Trailblazer cells that is consistent during migration and enriched for approximately 900 genes. Knockdown of several Trailblazer genes shows significant but modest changes to total distance migrated. However, in vivo expression analysis by RNAscope and immunohistochemistry reveals some salt and pepper patterns that include strong individual Trailblazer gene expression in cells within other subregions of the migratory stream. These data provide new insights into the molecular diversity and dynamics within a neural crest cell migratory stream that underlie complex directed and collective cell behaviors.

The architecture of single-stranded DNA-binding proteins, which play key roles in DNA metabolism, is based on different combinations of the oligonucleotide/oligosaccharide binding (OB) fold. Whereas the polypeptide serving this function in bacteria contains one OB fold, the eukaryotic functional homolog comprises a complex of three proteins, each harboring at least one OB fold. Here we show that unlike these groups of organisms, the Euryarchaeota has exploited the potential in the OB fold to re-invent single-stranded DNA-binding proteins many times. However, the most common form is a protein with two OB folds and one zinc finger domain. We created several deletion mutants of this protein based on its conserved motifs, and from these structures functional chimeras were synthesized, supporting the hypothesis that gene duplication and recombination could lead to novel functional forms of single-stranded DNAbinding proteins. Biophysical studies showed that the orthologs of the two OB fold/one zinc finger replication protein A in Methanosarcina acetivorans and Methanopyrus kandleri exhibit two binding modes, wrapping and stretching of DNA. However, the ortholog in Ferroplasma acidarmanus possessed only the stretching mode. Most interestingly, a second single-stranded DNAbinding protein, FacRPA2, in this archaeon exhibited the wrapping mode. Domain analysis of this protein, which contains a single OB fold, showed that its architecture is similar to the functional homologs thought to be unique to the Crenarchaeotes. Most unexpectedly, genes coding for similar proteins were found in the genomes of eukaryotes, including humans. Although the diversity shown by archaeal single-stranded DNA-binding proteins is unparalleled, the presence of their simplest form in many organisms across all domains of life is of greater evolutionary consequence.

SUMMARYNeural crest (NC) cells emerge from the dorsal trunk neural tube (NT) and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the NT have similar or differential ability to contribute to NC cells and their derivatives. To this end, we precisely labeled NT precursors at specific dorsoventral levels of the chick NT using fluorescent dyes and a photoconvertible fluorescent protein. NT and NC cell dynamics were then examined in vivo and in slice culture using two-photon and confocal time-lapse imaging. The results show that NC precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the NT, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the NT to contribute to NC derivatives, with the exception of sympathetic ganglia, which appeared to be 'filled' by the first population to emigrate. Rather than restricted developmental potential, however, this is probably due to a matter of timing.