Specific gene silencing using small interfering RNAs in fish embryos

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.     

Presence of serotonin in early chick embryos

With biochemical analysis and with autoradiography based on injection of 5-[3H]hydroxytryptophan, it was possible to demonstrate the presence of serotonin (5-hydroxytryptamine) in early chick embryos as early as the pre-streak stage. The biochemical analysis which covered the early developmental period (0.5-6 days of incubation) revealed an elevated concentration of serotonin at gastrulation; from then it stayed at a lower and fairly even level. Autoradiographs of embryos at the pre-streak stage, the primitive streak stage, the head fold stage and the 4-6 somites stage indicated the presence of serotonin in intracellular yolk granules and in cell nuclei. Moreover, the amine appeared associated with microfilaments and microtubules, particularly in developing neural cells. Notably the elevated concentration of serotonin at gastrulation, but also the intracellular distribution of the amine during early organogenesis, indicates a prominent role for it in cell-shape changes and morphogenesis in the early chick embryo.     

Rous sarcoma virus does not induce sarcoma in early chick embryos by lack of the v-src gene expression.

The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.     

Spatial and temporal 'knock down' of gene expression by electroporation of double-stranded RNA and morpholinos into early postimplantation mouse embryos

Here we report the use of double-stranded RNA (dsRNA) and morpholino technologies to specifically 'knock down' gene expression in early postimplantation mouse embryos. Sequence specific interference mediated by either dsRNA or by morpholino has been a useful tool for studying gene function in several organisms. However, specifically for the dsRNA, doubts have been raised about whether it could successfully be applied on vertebrate embryos. We demonstrate that electroporation of dsRNA directed against Otx2 or Foxa2 into postimplantation mouse embryos results in specific knock down of the expression of the respective endogenous genes in a region- and germ-layer specific manner. We also show that electroporation of morpholino directed against Foxa2 into the node of mouse embryos leads to a specific down regulation of Foxa2 expression in the floor plate. Our results demonstrate for the first time that dsRNA and morpholino technologies can be successfully applied in early postimplantation mouse embryos to specifically knock down gene expression.     

Cell junctions in explanted tissues from early chick embryos

Summary Hypoblast and definitive endoblast derived from young chick embryos were explanted and grown for 24 h in culture. The junctional complexes which characterise these tissues were studied on freeze-fracture replicas and thin sections. Cell membranes of the hypoblast displayed tight junctions only, disposed in randomly arranged strands or narrow belts which included many discontinuous strands. The definitive endoblast showed tight and gap junctions as well as desmosomes in close association with the tight junctions. It is suggested that the differences between the two types of tissue may be related to cell cohesiveness, which appears to be relatively low in the hypoblast and high in the definitive endoblast.     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.     

Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos.

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the most defining feature of chordates, and thus may be a key to understanding the origin and evolution of the chordates. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A recent study by [Tagawa et al. (1998)], however, revealed that a hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodaeum invagination region in the gastrula. The present study demonstrated that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos. Namely, ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region.     

Teratologic effects of LSD in explanted early chick embryos.

Chick embryos were explanted at stage; 4-7 and cultured for 20 h with or without LSD. At any stage 10 mug/ml LSD or higher caused abnormalities in axial structures, particularly somites, in over 50% of the embryos. LSD had no apparent effect on morphogenesis of the heart, but significantly lowered the pulse rate. Cellular degeneration occurred in severely affected structures, but LSD at embryotoxic doses caused alterations in neither cell morphology nor mitotic activity. The effects of LSD were not permanent, i.e., the embryos retained the ability to undergo normal morphogenesis when, after 4-5 h of treatment with 10 mug/ml LSD, they were subcultured on plain nutrient medium.     

Specific and nonspecific adhesion in the process of retinal cellular aggregation in chick embryos

A study of aggregation of the retinal cells of 8 and 14 day old chick embryos has revealed two phases in this process. The first phase includes the decrease in the concentration of single cells and the increase in the concentration of aggregates. During the second phase the concentration of aggregates falls at the expense of fusion of smaller aggregates into larger ones. The rate of aggregation at both these phases increases with the initial density of cells and decreases with the age of donor embryos and at a suboptimal temperature of cultivation. Aggregation during the first phase does not depend on the presence in the culture medium of divalent cations and colchicine, the level of protein and RNA synthesis in the cells, whereas aggregation during the second phase depends on all these factors. Comparison of these results with the published data suggests that the retinal cell aggregation during the second phase, unlike the first one, is based on the specific adhesiveness of the cells, which is realized via adhesion molecules resynthesized at the cell surface.     

Stage-specific gene expression in early chick embryo

Inhibition of DNA replication by aphidicolin in the chick morula interferes with its progression to a normal blastula and prevents induction of the first morphogenetic cell movements of primitive streak formation. Embryos in aphidicolin synthesize some polypeptides typical of blastula but do not display all the characteristic features of morula to blastula transition. Inhibition of DNA replication interferes with the sequential synthesis of maternally coded polypeptides and with the activation of the embryonic genome in the chick embryo.     

MAP kinase cascade,but not ERKs,activated during early cleavage of mouse embryos

Mitosis in early embryos is independent of exogenous mitogens, although mitogen stimulations and subsequent activation of a mitogen-activated protein (MAP) kinase cascade are essential for the proliferation of somatic cells. The activation state of the MAP kinase cascade during early cleavage has never been reported. In the present study, factors involved in the MAP kinase cascade—Ras, Raf-1, 14–3-3, MEK, and ERKs—and their activation states were detected by immunoblotting during early cleavage of mouse embryos. We found the constant presence of these molecules in mouse early embryos and the activation of Raf-1 exclusively at the M-phase. An immunoprecipitation study revealed that active Raf-1 in the M-phase was dissociated from 14–3-3, as in somatic cells, whereas inactive Raf-1 was associated with 14–3-3. Surprisingly, the ERKs (MAP kinases) were not activated throughout early cleavage, although M-phase–specific activation of the MAP kinase kinase, MEK was observed. Myelin basic protein kinase activity was, however, significantly higher in the M-phase than in the interphase. These results indicate that the MAP kinase cascade is activated at the M-phase and that some MAP kinases other than ERKs are activated during early cleavage of mouse embryos. Mol. Reprod. Dev. 51:148–155, 1998. © 1998 Wiley-Liss, Inc.     

Neural tube defects caused by local anesthetics in early chick embryos

The effects of local anesthetics (ketamine HCl, lidocaine HCl, procaine HCl, and tetracaine HCl) on stage 8 (four-somite) chick embryos were investigated. In general, embryos responded to drug treatment in a dose-related manner during the first 6 hr of incubation. Concentrations of 500 micrograms/ml (ca. 2 mM) or higher were embryolethal, whereas 100-200 micrograms/ml (0.1-0.8 mM) preferentially inhibited elevation of neural folds. The latter effect was detectable within 3 hr of treatment and was readily reversible. Tetracaine was the most potent among the four local anesthetics tested at any given dose. Compared to controls, cells in the defective neuroepithelium were less elongated and exhibited smoother apical (luminal) surfaces, thinner microfilament bundles, and less intense actin-specific fluorescence. Furthermore, the effects of local anesthetics (100-200 micrograms/ml) on stage 8 chick embryos were not identical to those of cytochalasin D (0.05 micrograms/ml), colchicine (1 microgram/ml), or ionophore A23187 (25 micrograms/ml), although all treatments produced neural tube defects. Overall results suggest that local anesthetics inhibit closure of the neural tube through their disruptive action on the organization and function of microfilaments in developing neuroepithelial cells.