In vivo dissection of Rhoa function in vascular development using zebrafish.

The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. In this study, we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant negative, or constitutively active forms of rhoaa in ECs, pharmacological inhibitors of RHOA and ROCK1/2, and Rock1 and Rock2a/b dgRNP-injected zebrafish embryos to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis in vivo, showing that either too much or too little RHOA activity leads to vascular dysfunction.

Targeted mutation of the gene encoding prion protein in zebrafish reveals a conserved role in neuron excitability.

The function of the cellular prion protein (PrP(C)) in healthy brains remains poorly understood, in part because Prnp knockout mice are viable. On the other hand, transient knockdown of Prnp homologs in zebrafish (including two paralogs, prp1 and prp2) has suggested that PrP(C) is required for CNS development, cell adhesion, and neuroprotection. It has been argued that zebrafish Prp2 is most similar to mammalian PrP(C), yet it has remained intransigent to the most thorough confirmations of reagent specificity during knockdown. Thus we investigated the role of prp2 using targeted gene disruption via zinc finger nucleases. Prp2(-/-) zebrafish were viable and did not display overt developmental phenotypes. Back-crossing female prp2(-/-) fish ruled out a role for maternal mRNA contributions. Prp2(-/-) larvae were found to have increased seizure-like behavior following exposure to the convulsant pentylenetetrazol (PTZ), as compared to wild type fish. In situ recordings from intact hindbrains demonstrated that prp2 regulates closing of N-Methyl-d-aspartate (NMDA) receptors, concomitant with neuroprotection during glutamate excitotoxicity. Overall, the knockout of Prp2 function in zebrafish independently confirmed hypothesized roles for PrP, identifying deeply conserved functions in post-developmental regulation of neuron excitability that are consequential to the etiology of prion and Alzheimer diseases.

Hmx4 regulates Sonic hedgehog signaling through control of retinoic acid synthesis during forebrain patterning.

Mutations in H6-homeobox (HMX) genes are linked to neural mispatterning and neural tube closure defects in humans. We demonstrate that zebrafish Hmx4 regulates the signaling of two morphogens critical for neural development, retinoic acid (RA) and Sonic hedgehog (Shh). Hmx4-depleted embryos have a strongly narrowed eye field and reduced forebrain Shh target gene expression. hmx4 morphants fail to properly transcribe the Shh signal transducer gli3, and have reduced ventral forebrain specification. Hmx4-depleted embryos also have neural tube patterning defects that phenocopy RA-deficiency. We show that Hmx4 is required for the initiation and maintenance of aldh1a2, the principal RA-synthesizing gene. Loss of RA is the primary defect in Hmx4-depleted embryos, as RA treatment rescues a number of the neural patterning defects. Surprisingly, RA treatment also rescues forebrain morphology, gli3 transcription, and Shh signaling. We propose that Hmx4 is a critical regulator of retinoic acid synthesis in a developing embryo, and that this regulation is essential for controlling Shh signaling and forebrain development.

Zebrafish Tshz3b negatively regulates Hox function in the developing hindbrain.

In flies, the zinc-finger protein Teashirt promotes trunk segmental identities, in part, by repressing the expression and function of anterior hox paralog group (PG) 1-4 genes that specify head fates. Anterior-posterior patterning of the vertebrate hindbrain also requires Hox PG 1-4 function, but the role of vertebrate teashirt-related genes in this process has not been investigated. In this work, we use overexpression and structure-function analyses to show that zebrafish tshz3b antagonizes Hox-dependent hindbrain segmentation. Ectopic Tshz3b perturbs the specification of rhombomere identities and leads to the caudal expansion of r1, the only rhombomere whose identity is specified independently of Hox function. This overexpression phenotype does not require the homeodomain and C-terminal zinc fingers that are unique to vertebrate Teashirt-related proteins, but does require that Tshz3b function as a repressor. Together, these results argue that the negative regulation of Hox PG 1-4 function is a conserved characteristic of Teashirt-related proteins.

The Hox cofactors Meis1 and Pbx act upstream of gata1 to regulate primitive hematopoiesis.

During vertebrate development, the initial wave of hematopoiesis produces cells that help to shape the developing circulatory system and oxygenate the early embryo. The differentiation of primitive erythroid and myeloid cells occurs within a short transitory period, and is subject to precise molecular regulation by a hierarchical cascade of transcription factors. The TALE-class homeodomain transcription factors Meis and Pbx function to regulate embryonic hematopoiesis, but it is not known where Meis and Pbx proteins participate in the hematopoietic transcription factor cascade. To address these questions, we have ablated Meis1 and Pbx proteins in zebrafish, and characterized their molecular effects on known markers of primitive hematopoiesis. Embryos lacking Meis1 and Pbx exhibit a severe reduction in the expression of gata1, the earliest marker of erythroid cell fate, and fail to produce visible circulating blood cells. Concomitant with a loss of gata1, Meis1- and Pbx-depleted embryos exhibit downregulated embryonic hemoglobin (hbae3) expression, and possess increased numbers of pu.1-positive myeloid cells. gata1-overexpression rescues hbae3 expression in Pbx-depleted; meis1-morphant embryos, placing Pbx and Meis1 upstream of gata1 in the erythropoietic transcription factor hierarchy. Our study conclusively demonstrates that Meis1 and Pbx act to specify the erythropoietic cell lineage and inhibit myelopoiesis.

The zebrafish: A fintastic model for hematopoietic development and disease.

Hematopoiesis is a complex process with a variety of different signaling pathways influencing every step of blood cell formation from the earliest precursors to final differentiated blood cell types. Formation of blood cells is crucial for survival. Blood cells carry oxygen, promote organ development and protect organs in different pathological conditions. Hematopoietic stem and progenitor cells (HSPCs) are responsible for generating all adult differentiated blood cells. Defects in HSPCs or their downstream lineages can lead to anemia and other hematological disorders including leukemia. The zebrafish has recently emerged as a powerful vertebrate model system to study hematopoiesis. The developmental processes and molecular mechanisms involved in zebrafish hematopoiesis are conserved with higher vertebrates, and the genetic and experimental accessibility of the fish and the optical transparency of its embryos and larvae make it ideal for in vivo analysis of hematopoietic development. Defects in zebrafish hematopoiesis reliably phenocopy human blood disorders, making it a highly attractive model system to screen small molecules to design therapeutic strategies. In this review, we summarize the key developmental processes and molecular mechanisms of zebrafish hematopoiesis. We also discuss recent findings highlighting the strengths of zebrafish as a model system for drug discovery against hematopoietic disorders. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion Vertebrate Organogenesis > Musculoskeletal and Vascular Nervous System Development > Vertebrates: Regional Development Comparative Development and Evolution > Organ System Comparisons Between Species.

Correction: Somite-Derived Retinoic Acid Regulates Zebrafish Hematopoietic Stem Cell Formation.

[This corrects the article DOI: 10.1371/journal.pone.0166040.].

Somite-Derived Retinoic Acid Regulates Zebrafish Hematopoietic Stem Cell Formation.

Hematopoietic stem cells (HSCs) are multipotent progenitors that generate all vertebrate adult blood lineages. Recent analyses have highlighted the importance of somite-derived signaling factors in regulating HSC specification and emergence from dorsal aorta hemogenic endothelium. However, these factors remain largely uncharacterized. We provide evidence that the vitamin A derivative retinoic acid (RA) functions as an essential regulator of zebrafish HSC formation. Temporal analyses indicate that RA is required for HSC gene expression prior to dorsal aorta formation, at a time when the predominant RA synthesis enzyme, aldh1a2, is strongly expressed within the paraxial mesoderm and somites. Previous research implicated the Cxcl12 chemokine and Notch signaling pathways in HSC formation. Consequently, to understand how RA regulates HSC gene expression, we surveyed the expression of components of these pathways in RA-depleted zebrafish embryos. During somitogenesis, RA-depleted embryos exhibit altered expression of jam1a and jam2a, which potentiate Notch signaling within nascent endothelial cells. RA-depleted embryos also exhibit a severe reduction in the expression of cxcr4a, the predominant Cxcl12b receptor. Furthermore, pharmacological inhibitors of RA synthesis and Cxcr4 signaling act in concert to reduce HSC formation. Our analyses demonstrate that somite-derived RA functions to regulate components of the Notch and Cxcl12 chemokine signaling pathways during HSC formation.

Evaluating the mutagenic activity of targeted endonucleases containing a Sharkey FokI cleavage domain variant in zebrafish.

Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.