Stephen C. Ekker

Effective targeted gene ‘knockdown’ in zebrafish

The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted ‘knockdown’ technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.

In vivo genome editing using a high-efficiency TALEN system

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

Patterning activities of vertebrate hedgehog proteins in the developing eye and brain

BACKGROUND The hedgehog (hh) family of secreted signaling proteins is responsible for developmental patterning in a variety of systems, including the neural tube, limbs and somites. Within the neural tube, at the level of the spinal cord, products of the vertebrate gene sonic hedgehog (shh) are proposed to function as a ventral patterning influence, with the capability of inducing floor plate and motor neurons. RESULTS We report the isolation of tiggy-winkle hedgehog (twhh), a novel member of the zebrafish hh gene family. Both twhh and shh are expressed in the ventral midline of the embryonic zebrafish neural tube and brain, but twhh expression becomes limited to the neural tube, whereas shh is also expressed in the notochord. Both genes are expressed in the developing brain, in domains that include a discrete region in the floor of the diencephalon, located between the sites of the future optic stalks. Using pax-2 and pax-6 as markers of proximo-distal fate within the developing eye, we found that ectopic expression of either hh gene promoted proximal fates and suppressed distal fates. In contrast, proximal fates were lost in cyclops mutant embryos, which lack twhh- and shh-expressing forebrain cells. Both twhh and shh proteins undergo autoproteolytic processing in vivo; a fragment corresponding to the amino-terminal cleavage product was sufficient to carry out all signaling activities associated with twhh in eye and brain development. CONCLUSIONS These findings suggest that secreted signals encoded by members of the hedgehog gene family, emanating from the ventral midline of the neural tube, not only play important roles in dorso-ventral patterning of the brain but also appear to constitute an early patterning activity along the proximo-distal axis of the developing eyes.

Hedgehog Patterning Activity: Role of a Lipophilic Modification Mediated by the Carboxy-Terminal Autoprocessing Domain

Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

Twisted is a conserved extracellular BMP antagonist

Bone morphogenetic protein (BMP) signalling regulates embryonic dorsal–ventral cell fate decisions in flies, frogs and fish. BMP activity is controlled by several secreted factors including the antagonists chordin and short gastrulation (SOG). Here we show that a second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signalling using anti-phospho-Smad staining shows that the tsg and sog loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, TSG and SOG together make a more effective inhibitor of BMP signalling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. We show that Tsgs from different species are functionally equivalent, and conclude that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signalling.

Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.

Distinct requirements for zebrafish angiogenesis revealed by a VEGF‐A morphant

Angiogenesis is a fundamental vertebrate developmental process that requires signalling by the secreted protein vascular endothelial growth factor‐A (VEGF‐A). VEGF‐A functions in the development of embryonic structures, during tissue remodelling and for the growth of tumour‐induced vasculature. The study of the role of VEGF‐A during normal development has been significantly complicated by the dominant, haplo‐insufficient nature of VEGF‐A‐targeted mutations in mice. We have used morpholino‐based targeted gene knock‐down technology to generate a zebrafish VEGF‐A morphant loss of function model. Zebrafish VEGF‐A morphant embryos develop with an enlarged pericardium and with major blood vessel deficiencies. Morphological assessment at 2 days of development indicates a nearly complete absence of both axial and intersegmental vasculature, with no or reduced numbers of circulating red blood cells. Molecular analysis using the endothelial markers fli‐1 and flk‐1 at 1 day of development demonstrates a fundamental distinction between VEGF‐A requirements for axial and intersegmental vascular structure specification. VEGF‐A is not required for the initial establishment of axial vasculature patterning, whereas all development of intersegmental vasculature is dependent on VEGF‐A signalling. The zebrafish thus serves as a quality model for the study of conserved vertebrate angiogenesis processes during embryonic development. Copyright

Mammalian germ-line transgenesis by transposition

Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA/transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.

The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins.

The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. We compare here the DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal‐B, binds preferentially to a sequence with an unusual 5′‐T‐T‐A‐T‐3′ core, whereas the other three prefer 5′‐T‐A‐A‐T‐3′. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity. We further show that amino acid residues within the N‐terminal arm are responsible for the sequence specificity differences between the Ultrabithorax and Abdominal‐B homeodomains. Similarities and differences at the corresponding positions within the N‐terminal arms are conserved in the vertebrate Abdominal‐B‐like HOM proteins, which play critical roles in limb specifications as well as in regional specification along the anterior‐posterior axis. This and other patterns of residue conservation suggest that differential DNA sequence recognition may play a role in HOM protein function in a wide range of organisms.

Mojo Hand, a TALEN design tool for genome editing applications

BackgroundRecent studies of transcription activator-like (TAL) effector domains fused to nucleases (TALENs) demonstrate enormous potential for genome editing. Effective design of TALENs requires a combination of selecting appropriate genetic features, finding pairs of binding sites based on a consensus sequence, and, in some cases, identifying endogenous restriction sites for downstream molecular genetic applications.ResultsWe present the web-based program Mojo Hand for designing TAL and TALEN constructs for genome editing applications (http://www.talendesign.org). We describe the algorithm and its implementation. The features of Mojo Hand include (1) automatic download of genomic data from the National Center for Biotechnology Information, (2) analysis of any DNA sequence to reveal pairs of binding sites based on a user-defined template, (3) selection of restriction-enzyme recognition sites in the spacer between the TAL monomer binding sites including options for the selection of restriction enzyme suppliers, and (4) output files designed for subsequent TALEN construction using the Golden Gate assembly method.ConclusionsMojo Hand enables the rapid identification of TAL binding sites for use in TALEN design. The assembly of TALEN constructs, is also simplified by using the TAL-site prediction program in conjunction with a spreadsheet management aid of reagent concentrations and TALEN formulation. Mojo Hand enables scientists to more rapidly deploy TALENs for genome editing applications.