We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. ...The donor construct was flanked with homology arms of about 1 kb at the 5' and 3' ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish.
Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can ...induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopu s genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F ₁ frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus .
Wnt/Frizzled (Fz) signaling controls cell polarity/movements during vertebrate gastrulation via incompletely defined mechanisms. We demonstrated previously that Wnt/Fz activation of Rho, a GTPase and ...regulator of cytoskeletal architecture, is essential for vertebrate gastrulation. Here we report that in mammalian cells and Xenopus embryos, Wnt/Fz signaling coactivates Rho and Rac, another GTPase and distinct regulator of cytoskeletal architecture. Wnt/Fz activation of Rac is independent of Rho and mediates Wnt/Fz activation of Jun N-terminal kinase (JNK). Dishevelled (Dvl), a cytoplasmic protein downstream of Fz, forms a Wnt-induced complex with Rac independent of the Wnt-induced Dvl-Rho complex. Depletion or inhibition of Rac function perturbs Xenopus gastrulation without affecting Wnt/Fz activation of the Rho or beta-catenin pathway. We propose that parallel activation of Rac and Rho pathways by Wnt/Fz signaling is required for cell polarity and movements during vertebrate gastrulation.
The neural crest develops in vertebrate embryos within a discrete domain at the neural plate boundary and eventually gives rise to a migrating population of cells that differentiate into a multitude ...of derivatives. We have shown that the broad-complex, tramtrack and bric a brac (BTB) domain-containing factor potassium channel tetramerization domain containing 15 (Kctd15) inhibits neural crest formation, and we proposed that its function is to delimit the neural crest domain. Here we report that Kctd15 is a highly effective inhibitor of transcription factor activating enhancer binding protein 2 (AP-2) in zebrafish embryos and in human cells; AP-2 is known to be critical for several steps of neural crest development. Kctd15 interacts with AP-2α but does not interfere with its nuclear localization or binding to cognate sites in the genome. Kctd15 binds specifically to the activation domain of AP-2α and efficiently inhibits transcriptional activation by a hybrid protein composed of the regulatory protein Gal4 DNA binding and AP-2α activation domains. Mutation of one proline residue in the activation domain to an alanine (P59A) yields a protein that is highly active but largely insensitive to Kctd15. These results indicate that Kctd15 acts in the embryo at least in part by specifically binding to the activation domain of AP-2α, thereby blocking the function of this critical factor in the neural crest induction hierarchy.
The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. ...Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation inlnx2adid not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition oflnx2aexon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.
Potassium channel tetramerization domain containing 15 (Kctd15) was previously found to have a role in early neural crest (NC) patterning, specifically delimiting the region where NC markers are ...expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs) to generate null mutations in zebrafish kctd15a and kctd15b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, kctd15a/b double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa), a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus kctd15 mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates.
Establishment of left-right asymmetry in vertebrates requires nodal, Wnt-PCP and FGF signaling and involves ciliogenesis in a laterality organ. Effector genes through which FGF signaling affects ...laterality have not been described. We isolated the zebrafish ier2 and fibp1 genes as FGF target genes and show that their protein products interact. Knock down of these factors interferes with establishment of organ laterality and causes defective cilia formation in Kupffer's Vesicle, the zebrafish laterality organ. Cilia are also lost after suppression of FGF8, but can be rescued by injection of ier2 and fibp1 mRNA. We conclude that Ier2 and Fibp1 mediate FGF signaling in ciliogenesis in Kupffer's Vesicle and in the establishment of laterality in the zebrafish embryo.
The Wnt–PCP (planar cell polarity, PCP) pathway regulates cell polarity and convergent extension movements during axis formation in vertebrates by activation of Rho and Rac, leading to the ...re‐organization of the actin cytoskeleton. Rho and Rac activation require guanine nucleotide‐exchange factors (GEFs), but the identity of the GEF involved in Wnt–PCP‐mediated convergent extension is unknown. Here we report the identification of the weak‐similarity GEF (WGEF) gene by a microarray‐based screen for notochord enriched genes, and show that WGEF is involved in Wnt‐regulated convergent extension. Overexpression of WGEF activated RhoA and rescued the suppression of convergent extension by dominant‐negative Wnt‐11, whereas depletion of WGEF led to suppression of convergent extension that could be rescued by RhoA or Rho‐associated kinase activation. WGEF protein preferentially localized at the plasma membrane, and Frizzled‐7 induced colocalization of Dishevelled and WGEF. WGEF protein can bind to Dishevelled and Daam‐1, and deletion of the Dishevelled‐binding domain generates a hyperactive from of WGEF. These results indicate that WGEF is a component of the Wnt–PCP pathway that connects Dishevelled to Rho activation.
Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These ...strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed beta-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased beta-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma.
Barx1 modulates cellular adhesion molecule expression and participates in specification of tooth-types, but little is understood of its role in patterning the pharyngeal arches. We examined
barx1 ...expression during zebrafish craniofacial development and performed a functional analysis using antisense morpholino oligonucleotides.
Barx1 is expressed in the rhombencephalic neural crest, the pharyngeal arches, the pectoral fin buds and the gut in contrast to its paralogue
barx2, which is most prominently expressed in the arch epithelium. Additionally,
barx1 transient expression was observed in the posterior lateral line ganglia and developing trunk/tail. We show that Barx1 is necessary for proliferation of the arch osteochondrogenic progenitors, and that morphants exhibit diminished and dysmorphic arch cartilage elements due to reductions in chondrocyte differentiation and condensation. Attenuation of Barx1 results in lost arch expression of osteochondrogenic markers
col2a1,
runx2a and
chondromodulin, as well as odontogenic marker
dlx2b. Further, loss of
barx1 positively influenced
gdf5 and
chordin, markers of jaw joint patterning. FGF signaling is required for maintaining
barx1 expression, and that ectopic BMP4 induces expression of
barx1 in the intermediate region of the second pharyngeal arch. Together, these results indicate an essential role for
barx1 at early stages of chondrogenesis within the developing zebrafish viscerocranium.
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