Expression of the zinc finger Egr1 gene during zebrafish embryonic development

Egr1 is a highly conserved zinc finger protein which plays important roles in many aspects of vertebrate development and in the adult. The cDNA coding for zebrafish Egr1 was obtained and its expression pattern was examined during zebrafish embryogenesis using whole-mount in situ hybridization. Egr1 mRNA is first detected in adaxial cells in the presomitic mesoderm between 11 and 20 h post-fertilization (hpf), spanning the 4-24 somite stages. Later, Egr1 expression is observed only in specific brain areas, starting at 21 hpf and subsequently increasing in distinct domains of the central nervous system, e.g. in the telencephalon, diencephalon and hypothalamus. Between 24 and 48 hpf, Egr1 is expressed in specific domains of the hypothalamus, mesencephalon, tegmentum, pharynx, retina, otic vesicle and heart.     

Targeted mutagenesis of zebrafish: use of zinc finger nucleases

The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.     

A zebrafish forebrain-specific zinc finger gene can induce ectopic dlx2 and dlx6 expression

Identification of the earliest forebrain-specific markers should facilitate the elucidation of molecular events underlying vertebrate forebrain determination and specification. Here we report the sequence and characterization of fez (forebrain embryonic zinc finger), a gene that is specifically expressed in the embryonic forebrain of zebrafish. Fez encodes a putative nuclear zinc finger protein that is highly conserved in Drosophila, zebrafish, Xenopus, mouse, and human. In zebrafish, the expression of fez becomes detectable at the anterior edge of the presumptive neuroectoderm by 70% epiboly. During the segmentation period, its expression is completely restricted to the rostral region of the prospective forebrain. At approximately 24 h postfertilization, fez expression is mostly confined to the telencephalon and the anterior-ventral region of the diencephalon. Although fez expression is present in one-eyed pinhead (oep) and cyclops (cyc) zebrafish mutants, the pattern is altered. Forced expression of fez induces ectopic expression of dlx2 and dlx6, two genes involved in brain development. Knockdown of fez function using a morpholino-based antisense oligo inhibited dlx2 expression in the ventral forebrain. Our studies indicate that fez is one of the earliest markers specific for the anterior neuroectoderm and it may play a role in forebrain development by regulating Dlx gene expression.     

Characterisation of duplicate zinc finger like 2 erythroid precursor genes in zebrafish

In separate expression pattern and micro-array screens the zinc finger containing factor, znfl2, has been previously implicated in hematopoiesis. Here we analysed znfl2 expression in detail and performed genetic epistatic analysis in a series of hematopoietic mutants and transient gain-of-function models. znfl2 expression in the hematopoietic intermediate mesoderm and derived erythrocytes required early genes cloche and spadetail, but not gata1. Expression was up-regulated in scl gain-of-function embryos, identifying znfl2 as an early erythroid factor that is regulated upstream or independently of gata1. Furthermore, we identified a duplicate znfl2 gene in the genome (znfl2b) which was expressed in early mesendoderm and weakly in the lateral plate mesoderm, overlapping in expression with znfl2. The production of loss-of-function models for znfl2, znfl2b and znfl2/znfl2b together suggested that these erythrocyte specific zinc finger genes are dispensible for erythropoiesis.     

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene

C₂H₂ zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C₂H₂ zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the -glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promotor:-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.     

Cloning and expression of a novel zinc finger gene, Fez, transcribed in the forebrain of Xenopus and mouse embryos

We have identified and cloned a novel zinc finger gene, Fez (forebrain embryonic zinc-finger), as a potential downstream determinant of anterior neural plate formation in Xenopus. Fez was isolated as one of several neural-specific genes that was induced by the neuralizing factor, noggin (Smith and Harland, 1992. Cell 70, 829-840), in uncommitted ectoderm. Fez has an open reading frame comprising 466 amino acids, and contains six C(2)H(2) type zinc finger domains, which are highly conserved among Drosophila, zebrafish, mouse, and human. In Xenopus, the expression of Fez begins at stage 12 in the rostral end of the neural plate, and by stage 45, it is localized to several telencephalic regions, including the olfactory bulbs, nervus terminalis, and ventricular zone. The mouse homologue of Fez is similarly expressed in the mouse forebrain by embryonic day 11.     

Phylogenetic analysis of the plant-specific zinc finger-homeobox and mini zinc finger gene families

Zinc finger-homeodomain proteins (ZHD) are present in many plants; however, the evolutionary history of the ZHD gene family remains largely unknown. We show here that ZHD genes are plant-specific, nearly all intronless, and related to MINI ZINC FINGER ( MIF ) genes that possess only the zinc finger. Phylogenetic analyses of ZHD genes from representative land plants suggest that non-seed plant ZHD genes occupy basal positions and angiosperm homologs form seven distinct clades. Several clades contain genes from two or more major angiosperm groups, including eudicots, monocots, magnoliids, and other basal angiosperms, indicating that several duplications occurred before the diversification of flowering plants. In addition, specific lineages have experienced more recent duplications. Unlike the ZHD genes, MIF s are found only from seed plants, possibly derived from ZHD s by loss of the homeodomain before the divergence of seed plants. Moreover, the MIF genes have also undergone relatively recent gene duplications. Finally, genome duplication might have contributed substantially to the expansion of family size in angiosperms and caused a high level of functional redundancy/overlap in these genes.     

Expression of a mouse zinc finger protein gene in both spermatocytes and oocytes during meiosis.

In order to identify genes regulating meiosis, a mouse spermatocyte cDNA library was screened for sequences encoding proteins with C2H2-type zinc finger motifs which are typically expressed by the Drosophila Krüppel gene. Three new cDNAs were isolated, and they were designated CTfin33, CTfin51, and CTfin92. Among them, CTfin51 was selected for further study. The deduced amino acid sequence revealed seven zinc finger motifs in its C-terminal region. Northern blot and in situ hybridization showed CTfin51 mRNA expression in spermatocytes after the pachytene stage and in early stage round spermatids of prepuberal and adult males. Immunocytochemical staining with an antiserum against beta-gal-CTfin51 fusion protein was localized within nuclei of spermatocytes and spermatids. Oocyte nuclei after the pachytene stage also were immunoreactive for CTfin51 protein. Immunoblots revealed a band at M(r) 75,000 in protein extracts from the testis and the ovary. These results suggest that the CTfin51 gene encodes a DNA-binding regulatory protein functionally associated with meiosis in both male and female gametogenesis.     

Expression of the zinc finger gene Gli3 is affected in the morphogenetic mouse mutant extra-toes (Xt).

Genetic analysis and homology between the phenotypic alterations of the human Greig Cephalopolysyndactyly Syndrome (GCPS) and the mouse mutant extra-toes (Xt) have suggested a dominant mutation in the same gene of both species. Recently, the GLI3 gene, a member of the Krüppel-related zinc finger genes, has been proposed as a candidate gene for GCPS. We examined the expression of the mouse Gli3 gene in both Xt mutant animals and during normal mouse development. Northern and RNAase protection analysis of embryos revealed that Gli3 expression was reduced about 50% in heterozygous Xt/+ mice and completely absent in homozygous Xt/Xt mice. In addition, in situ analysis of wild-type mice documented Gli3 expression in the developing limb and brain, structures affected in Xt mutant mice. This pattern suggests an important function of the Gli3 gene during morphogenesis.     

Expression pattern of the single-minded gene in zebrafish embryos.

Recently we isolated a homolog of the Drosophila single-minded (sim) gene from a zebrafish cDNA library. The 4380-bp of zebrafish sim cDNA encodes a polypeptide of 585 amino acids with strikingly conserved bHLH and PAS A/B domains in the amino-terminal region. During embryogenesis, sim mRNA appears in the animal hemisphere as early as 3 h post-fertilization and is expressed in a widespread pattern throughout the epiblast at the 75% epiboly stage. During the segmentation stage, sim mRNA is prominently expressed in the primordium of the hindbrain and appears as a transverse stripe in the epithelial layers of the mid-diencephalic boundary (MDB). During the pharyngula stage, sim is no longer expressed in the hindbrain, but continues to be expressed in the MDB and extends to the caudal diencephalon along the ventral midline. In addition, sim mRNA is prominent in the two pharyngeal arches. During the larval stage, sim mRNA is transcribed in the esophagus, liver, pancreas, and intestine. In contrast, sim mRNA is no longer detectable in the forebrain after hatching. In adult fish, sim is widely expressed in brain, eyes, gill, heart, liver, and intestine.     

Differential expression of the zinc finger gene Zfp105 during spermatogenesis

We report the isolation of Zfp105, the mouse homolog of the human ZNF35 zinc finger gene. Zfp105 and ZNF35 are highly conserved at the protein and nucleotide level, and Zfp105 maps to a region of mouse Chromosome (Chr) 9 that is homologous to the human region containing ZNF35. Zpf105 is highly expressed in the testis, especially in pachytene spermatocytes and round spermatids. The possible role of this gene product in maintaining an ordered germ cell differentiation process is discussed. Received: 11 February 1998 / Accepted: 1 May 1998     

Expression, purification and characterization of cloning-grade zinc finger nuclease

The limited number of naturally occurring rare-cutting restriction enzymes and the slow and tedious engineering of existing restriction enzymes for novel specificities have prompted the design of new strategies for the development of restriction enzymes with specificities for long DNA sequences. One possibility is using zinc finger nucleases (ZFNs)—synthetic restriction enzymes that are custom-designed to target and cleave long DNA sequences and which have been recently shown useful for DNA cloning. Here we report on the purification and biochemical analysis of ZFN-10, a custom-made ZFN. We show that Ni-affinity and gel-filtration purification methods are sufficient to produce a cloning-grade enzyme. We show that ZFN-10 can function as an accurate and reliable ZFN using the same reagents and protocols used for naturally occurring and commercially available recombinant restriction enzymes. We also show that ZFN-10 tolerates a set of target-site substitutions which can be predicted from the specificities of recognition helices incorporated into the structure of its DNA-binding domain. The relative simplicity of ZFN-10 design, expression, purification and analysis suggests that novel ZFNs can potentially be designed and applied for various recombinant DNA applications.     

Expression of the chaperonin 10 gene during zebrafish development

We have isolated a cDNA encoding chaperonin 10 (cpn10) from the zebrafish. Using northern, western, and in situ hybridization analysis, we observed that the cpn10 gene is expressed uniformly and ubiquitously throughout embryonic development of the zebrafish. Upregulation of cpn10 expression was observed following exposure of zebrafish embryos to a heat shock of 1 hour at 37 degrees C compared to control embryos raised at 27 degrees C. The extracellular form of Cpn10 called early pregnancy factor (EPF), found in the serum of pregnant mammals, was not detected in the serum of either male or female zebrafish. These expression studies suggest that Cpn10 plays a general role in zebrafish development as well as being consistent with the hypothesis that EPF is involved in the embryo implantation process in mammals.     

Regulatory gene expression patterns reveal transverse and longitudinal subdivisions of the embryonic zebrafish forebrain

To shed light on the organization of the rostral embryonic brain of a lower vertebrate, we have directly compared the expression patterns of dlx, fgf, hh, hlx, otx, pax, POU, winged helix and wnt gene family members in the fore- and midbrain of the zebrafish. We show that the analyzed genes are expressed in distinct transverse and longitudinal domains and share expression boundaries at stereotypic positions within the fore- and midbrain. Some of these shared expression boundaries coincide with morphological landmarks like the pathways of primary axon tracts. We identified a series of eight transverse diencephalic domains suggestive of neuromeric subdivisions within the rostral brain. In addition, we identified four molecularly distinct longitudinal subdivisions and provide evidence for a strong bending of the longitudinal rostral brain axis at the cephalic flexure. Our data suggest a strong conservation of early forebrain organization between lower and higher vertebrates.