We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.

Early embryonic development in many organisms relies upon maternal molecules deposited into the egg prior to fertilization. We have cloned and characterized a maternal T-box gene in the zebrafish, eomesodermin(eomes). During oogenesis, the eomes transcript becomes localized to the cortex of the oocyte. After fertilization during early cleavage stages, eomes is expressed in a vegetal to animal gradient in the embryo, whereas Eomesodermin protein (Eom) is distributed cytoplasmically throughout the blastoderm. Strikingly, following midblastula transition, nuclear-localized Eomesodermin is detected on the dorsal side of the embryo only. Overexpression of eomes results in Nodal-dependent and nieuwkoid/dharma (nwk/dhm) independent ectopic expression of the organizer markers goosecoid (gsc), chordin (chd) and floating head (flh) and in the formation of secondary axes. The same phenotypes are observed when a VP16-activator construct is injected into early embryos, indicating that eomes acts as a transcriptional activator. In addition, a dominant-negative construct and antisense morpholino oligonucleotides led to a reduction in gsc and flh expression. Together these data indicate that eomes plays a role in specifying the organizer.

Gastrulation involves of a series of coordinated cell movements to organize the germ layers and establish the major body axes of the embryo. One gastrulation movement is epiboly, which involves the thinning and spreading of a multilayered cell sheet. Epiboly plays a prominent role in zebrafish gastrulation and studies of zebrafish epiboly have provided insights into basic cellular properties and mechanisms of morphogenesis that are widely used in animal development. Although considerable progress has been made in identifying molecules that are required for epiboly, we still understand very little about how these factors cooperate to drive the process. Here, we review work on the molecular and cellular basis of zebrafish epiboly in order to identify unifying themes and to highlight some of the current open questions.

In response to a pulling force, a material can elongate, hold fast, or fracture. During animal development, multi-cellular contraction of one region often stretches neighboring tissue. Such local contraction occurs by induced actomyosin activity, but molecular mechanisms are unknown for regulating the physical properties of connected tissue for elongation under stress. We show that cytohesins, and their Arf small G protein guanine nucleotide exchange activity, are required for tissues to elongate under stress during both Drosophila dorsal closure (DC) and zebrafish epiboly. In Drosophila, protein localization, laser ablation, and genetic interaction studies indicate that the cytohesin Steppke reduces tissue tension by inhibiting actomyosin activity at adherens junctions. Without Steppke, embryogenesis fails, with epidermal distortions and tears resulting from myosin misregulation. Remarkably, actomyosin network assembly is necessary and sufficient for local Steppke accumulation, where live imaging shows Steppke recruitment within minutes. This rapid negative feedback loop provides a molecular mechanism for attenuating the main tension generator of animal tissues. Such attenuation relaxes tissues and allows orderly elongation under stress.