Masahiro Shin

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.

Targeted chromosomal deletions and inversions in zebrafish

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

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.

Distinct Notch signaling outputs pattern the developing arterial system

Differentiation of arteries and veins is essential for the development of a functional circulatory system. In vertebrate embryos, genetic manipulation of Notch signaling has demonstrated the importance of this pathway in driving artery endothelial cell differentiation. However, when and where Notch activation occurs to affect endothelial cell fate is less clear. Using transgenic zebrafish bearing a Notch-responsive reporter, we demonstrate that Notch is activated in endothelial progenitors during vasculogenesis prior to blood vessel morphogenesis and is maintained in arterial endothelial cells throughout larval stages. Furthermore, we find that endothelial progenitors in which Notch is activated are committed to a dorsal aorta fate. Interestingly, some arterial endothelial cells subsequently downregulate Notch signaling and then contribute to veins during vascular remodeling. Lineage analysis, together with perturbation of both Notch receptor and ligand function, further suggests several distinct developmental windows in which Notch signaling acts to promote artery commitment and maintenance. Together, these findings demonstrate that Notch acts in distinct contexts to initiate and maintain artery identity during embryogenesis.

Notch mediates Wnt and BMP signals in the early separation of smooth muscle progenitors and blood/endothelial common progenitors

During embryonic development in amniotes, the extraembryonic mesoderm, where the earliest hematopoiesis and vasculogenesis take place, also generates smooth muscle cells (SMCs). It is not well understood how the differentiation of SMCs is linked to that of blood (BCs) and endothelial (ECs) cells. Here we show that, in the chick embryo, the SMC lineage is marked by the expression of a bHLH transcription factor, dHand. Notch activity in nascent ventral mesoderm cells promotes SMC progenitor formation and mediates the separation of SMC and BC/EC common progenitors marked by another bHLH factor, Scl. This is achieved by crosstalk with the BMP and Wnt pathways, which are involved in mesoderm ventralization and SMC lineage induction, respectively. Our findings reveal a novel role of the Notch pathway in early ventral mesoderm differentiation, and suggest a stepwise separation among its three main lineages, first between SMC progenitors and BC/EC common progenitors, and then between BCs and ECs.

Activin/TGF-beta signaling regulates Nanog expression in the epiblast during gastrulation.

Nanog is required for the maintenance of cellular pluripotency during normal development and in cultured embryonic stem cells. A number of signaling pathways have been implicated in regulating Nanog gene expression in vitro. Using the chick model, we provide in vivo evidence for the involvement of the Activin/TGF-beta signaling pathway in regulating Nanog expression in epiblast cells during gastrulation. Nanog expression in primordial germ cells is not regulated by this pathway, indicating that these two cell types employ different mechanisms for maintaining pluripotency in early development. Furthermore, our data suggest that the bHLH factor E2A plays a role in negatively regulating Nanog expression in vivo. Overall, our data support a direct and positive role of the Smad2/3 mediated TGF-beta signaling pathway in inducing/maintaining Nanog expression.

Venous-derived angioblasts generate organ-specific vessels during zebrafish embryonic development.

Formation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. Although it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels and the molecular mechanisms regulating their formation are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas and, as such, represents an ideal model in which to investigate the early steps of organ-specific vessel formation. Here, we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the posterior cardinal vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein forms and expands ventrally through a Bone Morphogenetic Protein-dependent step of collective migration. Concomitantly, a Vascular Endothelial Growth Factor-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers, is observed, which culminates with the generation of the supraintestinal artery. Together, our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late-forming, tissue-specific vascular beds. Highlighted article: A specialized pool of angioblasts is the origin of the zebrafish subintestinal plexus, a structure that gives rise to the organ-specific vessels of the gut, liver and pancreas.

Vegfa signals through ERK to promote angiogenesis, but not artery differentiation

Vascular endothelial growth factor a (Vegfa) is essential for blood vessel formation and can induce activation of numerous signaling effectors in endothelial cells. However, it is unclear how and where these function in developmental contexts during vascular morphogenesis. To address this issue, we have visualized activation of presumptive Vegfa effectors at single-cell resolution in zebrafish blood vessels. From these studies, we find that phosphorylation of the serine/threonine kinase ERK (pERK) preferentially occurs in endothelial cells undergoing angiogenesis, but not in committed arterial endothelial cells. pERK in endothelial cells was ectopically induced by Vegfa and lost in Vegfa signaling mutants. Both chemical and endothelial autonomous inhibition of ERK prevented endothelial sprouting, but did not prevent initial artery differentiation. Timed chemical inhibition during angiogenesis caused a loss of genes implicated in coordinating tip/stalk cell behaviors, including flt4 and, at later stages, dll4. ERK inhibition also blocked excessive angiogenesis and ectopic flt4 expression in Notch-deficient blood vessels. Together, these studies implicate ERK as a specific effector of Vegfa signaling in the induction of angiogenic genes during sprouting. Summary: ERK acts as a specific effector of Vegfa signaling to induce angiogenic genes during vascular sprouting in zebrafish.

BetaA, the major beta globin in definitive red blood cells, is present from the onset of primitive erythropoiesis in chicken

Reflecting physiological changes in oxygen acquisition and regulatory changes in globin transcription, the makeup of globin chains in erythrocytes varies in development and disease. The relationship between the globin chain composition and erythropoietic lineages/niches is not well‐understood. Using a combination of proteomic‐, genomic‐, and intron‐based in situ hybridization analyses, we show that the transcripts and protein product of the major adult beta globin, betaA, are present as early as the major embryonic beta globins during chicken primitive erythropoiesis. A rapid rise in betaA percentage is seen from embryonic day (E) 5, reaching adult profile by E7. Our data suggest that betaA locus is active from the onset of primitive erythropoiesis and that beta globin switching during development may reflect a change in relative transcript abundance rather than a strict on/off switch in gene activation. Developmental Dynamics 237:1193–1197, 2008.

Identification, characterization, and expression pattern of the chicken EKLF gene

EKLF/KLF1 was the first of the Krüppel‐like factors (KLFs) to be identified in mammals and plays an important role in primitive and definitive erythropoiesis. Here, we identify and characterize EKLF in the chicken (cEKLF). The predicted amino acid sequence of the zinc finger region of cEKLF is at least 87.7% similar to mammalian EKLF proteins and is 98.8% and 95% similar to the EKLF orthologues in Xenopus and zebrafish, respectively. During early embryonic development, cEKLF expression is seen in the posterior primitive streak, which gives rise to hematopoietic cells, and then in the blood islands and in circulating blood cells. cEKLF mRNA is expressed in blood cells but not in brain later in chicken embryonic development. cEKLF mRNA is increased in definitive compared with primitive erythropoiesis. The conserved sequence and expression pattern of cEKLF suggests that its function is similar to its orthologues in mammals, Xenopus, and zebrafish. Developmental Dynamics 235:1933–1940, 2006.