Generation and characterization of Kctd15 mutations in zebrafish.

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.

PCR artifact in testing for homologous recombination in genomic editing in zebrafish.

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.

Genes regulated by potassium channel tetramerization domain containing 15 (Kctd15) in the developing neural crest.

Neural crest (NC) development is controlled precisely by a regulatory network with multiple signaling pathways and the involvement of many genes. The integration and coordination of these factors are still incompletely understood. Overexpression of Wnt3a and the BMP antagonist Chordin in animal cap cells from Xenopus blastulae induces a large number of NC specific genes. We previously suggested that Potassium Channel Tetramerization Domain containing 15 (Kctd15) regulates NC formation by affecting Wnt signaling and the activity of transcription factor AP-2. In order to advance understanding of the function of Kctd15 during NC development, we performed DNA microarray assays in explants injected with Wnt3a and Chordin, and identified genes that are affected by Kctd15 overexpression. Among the many genes identified, we chose Duf domain containing protein 1 (ddcp1), Platelet-Derived Growth Factor Receptor a (pdgfra), Complement factor properdin (cfp), Zinc Finger SWIM-Type Containing 5 (zswim5), and complement component 3 (C3) to examine their expression by whole mount in situ hybridization. Our work points to a possible role for Kctd15 in the regulation of NC formation and other steps in embryonic development.

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.

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 in lnx2a did 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 of lnx2a exon 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.

Custos controls ß-catenin to regulate head development during vertebrate embryogenesis.

Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of ß-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited ß-catenin/TCF-dependent transcription. Custos binds to ß-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of ß-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-ß-catenin signal transduction cascade.

Cell adhesion in zebrafish embryos is modulated by March 8.

March 8 is a member of a family of transmembrane E3 ubiquitin ligases that have been studied mostly for their role in the immune system. We find that March 8 is expressed in the zebrafish egg and early embryo, suggesting a role in development. Both knock-down and overexpression of March 8 leads to abnormal development. The phenotype of zebrafish embryos and Xenopus animal explants overexpressing March 8 implicates impairment of cell adhesion as a cause of the effect. In zebrafish embryos and in cultured cells, overexpression of March 8 leads to a reduction in the surface levels of E-cadherin, a major cell-cell adhesion molecule. Experiments in cell culture further show that E-cadherin can be ubiquitinated by March 8. On the basis of these observations we suggest that March 8 functions in the embryo to modulate the strength of cell adhesion by regulating the localization of E-cadherin.

The BTB-containing protein Kctd15 is SUMOylated in vivo.

Potassium Channel Tetramerization Domain containing 15 (Kctd15) has a role in regulating the neural crest (NC) domain in the embryo. Kctd15 inhibits NC induction by antagonizing Wnt signaling and by interaction with the transcription factor AP-2α activation domain blocking its activity. Here we demonstrate that Kctd15 is SUMOylated by SUMO1 and SUMO2/3. Kctd15 contains a classical SUMO interacting motif, ψKxE, at the C-terminal end, and variants of the motif within the molecule. Kctd15 SUMOylation occurs exclusively in the C-terminal motif. Inability to be SUMOylated did not affect Kctd15's subcellular localization, or its ability to repress AP-2 transcriptional activity and to inhibit NC formation in zebrafish embryos. In contrast, a fusion of Kctd15 and SUMO had little effectiveness in AP-2 inhibition and in blocking of NC formation. These data suggest that the non-SUMOylated form of Kctd15 functions in NC development.

Habenular commissure formation in zebrafish is regulated by the pineal gland-specific gene unc119c.

BACKGROUND: The zebrafish pineal gland (epiphysis) is a site of melatonin production, contains photoreceptor cells, and functions as a circadian clock pacemaker. Since it is located on the surface of the forebrain, it is accessible for manipulation and, therefore, is a useful model system to analyze pineal gland function and development. We previously analyzed the pineal transcriptome during development and showed that many genes exhibit a highly dynamic expression pattern in the pineal gland. RESULTS: Among genes preferentially expressed in the zebrafish pineal gland, we identified a tissue-specific form of the unc119 gene family, unc119c, which is highly preferentially expressed in the pineal gland during day and night at all stages examined from embryo to adult. When expression of unc119c was inhibited, the formation of the habenular commissure (HC) was specifically compromised. The Unc119c interacting factors Arl3l1 and Arl3l2 as well as Wnt4a also proved indispensible for HC formation. CONCLUSIONS: We suggest that Unc119c, together with Arl3l1/2, plays an important role in modulating Wnt4a production and secretion during HC formation in the forebrain of the zebrafish embryo.

Inhibition of neural crest formation by Kctd15 involves regulation of transcription factor AP-2.

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.

Novel oxytocin gene expression in the hindbrain is induced by alcohol exposure: transgenic zebrafish enable visualization of sensitive neurons.

BACKGROUND: Fetal Alcohol Spectrum Disorders (FASD) are a collection of disorders resulting from fetal ethanol exposure, which causes a wide range of physical, neurological and behavioral deficits including heightened susceptibility for alcoholism and addictive disorders. While a number of mechanisms have been proposed for how ethanol exposure disrupts brain development, with selective groups of neurons undergoing reduced proliferation, dysfunction and death, the induction of a new neurotransmitter phenotype by ethanol exposure has not yet been reported. PRINCIPAL FINDINGS: The effects of embryonic and larval ethanol exposure on brain development were visually monitored using transgenic zebrafish expressing cell-specific green fluorescent protein (GFP) marker genes. Specific subsets of GFP-expressing neurons were highly sensitive to ethanol exposure, but only during defined developmental windows. In the med12 mutant, which affects the Mediator co-activator complex component Med12, exposure to lower concentrations of ethanol was sufficient to reduce GFP expression in transgenic embryos. In transgenic embryos and larva containing GFP driven by an oxytocin-like (oxtl) promoter, ethanol exposure dramatically up-regulated GFP expression in a small group of hindbrain neurons, while having no effect on expression in the neuroendocrine preoptic area. CONCLUSIONS: Alcohol exposure during limited embryonic periods impedes the development of specific, identifiable groups of neurons, and the med12 mutation sensitizes these neurons to the deleterious effects of ethanol. In contrast, ethanol exposure induces oxtl expression in the hindbrain, a finding with profound implications for understanding alcoholism and other addictive disorders.

Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).

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 Xenopus 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.

Homeoprotein hhex-induced conversion of intestinal to ventral pancreatic precursors results in the formation of giant pancreata in Xenopus embryos.

Liver and ventral pancreas develop from neighboring territories within the endoderm of gastrulae. ventral pancreatic precursor 1 (vpp1) is a marker gene that is differentially expressed in a cell population within the dorsal endoderm in a pattern partially overlapping with that of hematopoietically expressed homeobox (hhex) during gastrulation. In tail bud embryos, vpp1 expression specifically demarcates two ventral pancreatic buds, whereas hhex expression is mainly restricted to the liver diverticulum. Ectopic expression of a critical dose of hhex led to a greatly enlarged vpp1-positive domain and, subsequently, to the formation of giant ventral pancreata, putatively by conversion of intestinal to ventral pancreatic precursor cells. Conversely, antisense morpholino oligonucleotide-mediated knockdown of hhex resulted in a down-regulation of vpp1 expression and a specific loss of the ventral pancreas. Furthermore, titration of hhex with a dexamethasone-inducible hhex-VP16GR fusion construct suggested that endogenous hhex activity during gastrulation is essential for the formation of ventral pancreatic progenitor cells. These observations suggest that, beyond its role in liver development, hhex controls specification of a vpp1-positive endodermal cell population during gastrulation that is required for the formation of the ventral pancreas.

Vap (Vascular Associated Protein): a novel factor involved in erythropoiesis and angiogenesis.

Both endothelial and erythroid cells are generated in the intermediate cell mass (ICM) during zebrafish embryogenesis, but the nature of the genes that contribute to the processes of erythrocyte maturation and blood vessel network formation is not fully understood. From our in situ-based screening, we have identified a novel factor, Vap (Vascular Associated Protein) that is predominantly expressed in the ICM, and subsequently enriched in endothelial cells. Vap expression in the ICM was drastically suppressed in the cloche mutant that has defects in both vasculogenesis and hematopoiesis, whereas Vap expression was not affected in the vlad tepes/gata1 mutant. Knockdown of Vap using anti-sense morpholinos (VAP-MO) not only resulted in decreased numbers of erythrocytes but also in the strong suppression of hemoglobin production. Further, we found that Vap knockdown caused the disorganization of the intersegmental vessels (ISVs), which show irregular branching. We propose that Vap plays an important role in the maturation of endothelial and erythroid cells in zebrafish.

Solute carrier family 3 member 2 (Slc3a2) controls yolk syncytial layer (YSL) formation by regulating microtubule networks in the zebrafish embryo.

The yolk syncytial layer (YSL) in the zebrafish embryo is a multinucleated syncytium essential for embryo development, but the molecular mechanisms underlying YSL formation remain largely unknown. Here we show that zebrafish solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL and that slc3a2 knockdown causes severe YSL defects including clustering of the yolk syncytial nuclei and enhanced cell fusion, accompanied by disruption of microtubule networks. Expression of a constitutively active RhoA mimics the YSL phenotypes caused by slc3a2 knockdown, whereas attenuation of RhoA or ROCK activity rescues the slc3a2-knockdown phenotypes. Furthermore, slc3a2 knockdown significantly reduces tyrosine phosphorylation of c-Src, and overexpression of a constitutively active Src restores the slc3a2-knockdown phenotypes. Our data demonstrate a signaling pathway regulating YSL formation in which Slc3a2 inhibits the RhoA/ROCK pathway via phosphorylation of c-Src to modulate YSL microtubule dynamics. This work illuminates processes at a very early stage of zebrafish embryogenesis and more generally informs the mechanism of cell dynamics during syncytium formation.

Modulation of Tcf3 repressor complex composition regulates cdx4 expression in zebrafish.

The caudal homeobox (cdx) gene family is critical for specification of caudal body formation and erythropoiesis. In zebrafish, cdx4 expression is controlled by the Wnt pathway, but the molecular mechanism of this regulation is not fully understood. Here, we provide evidence that Tcf3 suppresses cdx4 expression through direct binding to multiple sites in the cdx4 gene regulatory region. Tcf3 requires corepressor molecules such as Groucho (Gro)/TLE and HDAC1 for activity. Using zebrafish embryos and cultured mammalian cells, we show that the transcription factor E4f1 derepresses cdx4 by dissociating corepressor proteins from Tcf3 without inhibiting its binding to cis-regulatory sites in the DNA. Further, the E3 ubiquitin ligase Lnx2b, acting as a scaffold protein irrespective of its enzymatic activity, counteracts the effects of E4f1. We propose that the modulation of Tcf3 repressor function by E4f1 assures precise and robust regulation of cdx4 expression in the caudal domain of the embryo.

The transcriptional mediator component Med12 is required for hindbrain boundary formation.

BACKGROUND: Rhombomere boundaries form during hindbrain segmentation and are critical for maintaining segmental integrity and regulating migration in the hindbrain. Some genetic models affecting hindbrain boundary formation have been described, but involvement of components of the transcriptional mediator complex in boundary formation has not reported so far. PRINCIPAL FINDINGS: The kto/med12 mutant zebrafish, which affects the Mediator component Med12, causes specific loss of rhombomere boundary cells even though segmentation of the hindbrain takes place at least in part. In kto mutant embryos, cells forming rhombomere boundaries were largely absent as indicated by the use of several marker genes. While no obvious increase in cell death was observed, we found a notable reduction of cell proliferation in the hindbrain of kto mutant zebrafish. CONCLUSIONS: The kto/med12 mutation results in specific defects of boundary cell formation in the zebrafish hindbrain.

Lhx1 is required for specification of the renal progenitor cell field.

In the vertebrate embryo, the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. In this study, we investigated the role of Lhx1 in specification of the kidney field by either overexpressing or depleting lhx1 in Xenopus embryos or depleting lhx1 in an explant culture system. By overexpressing a constitutively-active form of Lhx1, we established its capacity to expand the kidney field during the specification stage of kidney organogenesis. In addition, the ability of Lhx1 to expand the kidney field diminishes as kidney organogenesis transitions to the morphogenesis stage. In a complimentary set of experiments, we determined that embryos depleted of lhx1, show an almost complete loss of the kidney field. Using an explant culture system to induce kidney tissue, we confirmed that expression of genes from both proximal and distal kidney structures is affected by the absence of lhx1. Taken together our results demonstrate an essential role for Lhx1 in driving specification of the entire kidney field from the intermediate mesoderm.

XIer2 is required for convergent extension movements during Xenopus development.

Immediate early response 2 (Ier2) is a downstream target of fibroblast growth factor (FGF) signaling. In zebrafish, Ier2 is involved in left-right asymmetry establishment and in convergent extension movements. We isolated the Xenopus ier2 gene based on sequence similarity searches using multiple vertebrate species. Xenopus Ier2 has high homology in the N-terminal region to other vertebrate Ier2 proteins, and Xier2 transcripts were observed from oocytes through larval stages. Except for the maternal expression of xier2, the expression of this gene in the marginal region at gastrulation and in somites and the notochord at later stages is similar to the expression pattern of zebrafish ier2. XIer2 knockdown using antisense morpholinos resulted in defects of convergent extension leading to severe neural tube defects; overexpression of Ier2 showed similar, albeit milder phenotypes. Assays in animal cap explants likewise showed inhibition of elongation after blocking XIer2 expression. These results indicate that Xenopus Ier2 is essential for the execution of convergent extension movements during early Xenopus development.

Cloning and developmental expression of zebrafish pdzrn3.

Pdzrn3, a member of the PDZRN/SEMCAP/LNX protein family containing a RING finger and two PDZ domains, has been implicated in myoblast and osteoblast differentiation. However, its spatio-temporal expression pattern during embryonic development has not been defined. Here, we describe the cloning and expression pattern of pdzrn3 during zebrafish development. We found that in addition to being expressed in several mesodermal structures, this gene displays specific expression in the central nervous system including rhombomere 1, ventral retina, thalamus and motor neurons, indicating a novel function during neural development. In particular, the absence of expression of pdzrn3 in the ventral retina of noi mutant fish suggests a possible role for this gene in regulating fasciculation and/or navigation of retinal ganglion cell axons.