cyclops encodes a nodal-related factor involved in midline signaling.

Ventral structures in the central nervous system are patterned by signals emanating from the underlying mesoderm as well as originating within the neuroectoderm. Mutations in the zebrafish, Danio rerio, are proving instrumental in dissecting these midline signals. The cyclops mutation leads to a loss of medial floor plate and to severe deficits in ventral forebrain development, leading to cyclopia. Here, we report that the cyclops locus encodes the nodal-related protein Ndr2, a member of the transforming growth factor type beta superfamily of factors. The evidence includes identification of a missense mutation in the initiation codon and rescue of the cyclops phenotype by expression of ndr2 RNA, here renamed "cyclops." Thus, in interaction with other molecules implicated in these processes such as sonic hedgehog and one-eyed-pinhead, cyclops is required for ventral midline patterning of the embryonic central nervous system.

Zebrafish nodal-related genes are implicated in axial patterning and establishing left-right asymmetry.

Nodal-related 1 (ndr1) and nodal-related 2 (ndr2) genes in zebrafish encode members of the nodal subgroup of the transforming growth factor-beta superfamily. We report the expression patterns and functional characteristics of these factors, implicating them in the establishment of dorsal-ventral polarity and left-right asymmetry. Ndr1 is expressed maternally, and ndr1 and ndr2 are expressed during blastula stage in the blastoderm margin. During gastrulation, ndr expression subdivides the shield into two domains: a small group of noninvoluting cells, the dorsal forerunner cells, express ndr1, while ndr2 RNA is found in the hypoblast layer of the shield and later in notochord, prechordal plate, and overlying anterior neurectoderm. During somitogenesis, ndr2 is expressed asymmetrically in the lateral plate as are nodal-related genes of other organisms, and in a small domain in the left diencephalon, providing the first observation of asymmetric gene expression in the embryonic forebrain. RNA injections into Xenopus animal caps showed that Ndr1 acts as a mesoderm inducer, whereas Ndr2 is an efficient neural but very inefficient mesoderm inducer. We suggest that Ndr1 has a role in mesoderm induction, while Ndr2 is involved in subsequent specification and patterning of the nervous system and establishment of laterality.

Expression of activin transcripts in follicle cells and oocytes of Xenopus laevis.

Activin is a potent inducer of axial mesoderm in vitro and may have a similar role in vivo. Xenopus laevis eggs contain significant amounts of activin or activin-like factors, but maternal activin transcripts have not been detected in Xenopus eggs. The maternal activin protein might be translated from activin beta A or beta B mRNAs that are transiently expressed during oogenesis, or activin polypeptides might be transferred from follicle cells to oocytes. To assess these possibilities we studied activin mRNAs in follicle cells and oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNA blotting. Activin beta A, beta B1, and beta B2 transcripts occur in follicle cells; among them, beta A mRNA is by far the most abundant. Activin beta A and beta B1 mRNAs were not detected by RT-PCR in the corresponding stage IV oocytes, but activin beta B2 transcripts were found at low levels. These observations are consistent with synthesis of activin beta A and possibly beta B polypeptides in follicle cells followed by their secretion and uptake by oocytes, although synthesis of activin beta B2 in the oocytes could make a contribution to the maternal activin pool.

The role of growth factors in embryonic induction in Xenopus laevis.

Establishment of the body pattern in all animals, and especially in vertebrate embryos, depends on cell interactions. During the cleavage and blastula stages in amphibians, signal(s) from the vegetal region induce the equatorial region to become mesoderm. Two types of peptide growth factors have been shown by explant culture experiments to be active in mesoderm induction. First, there are several isoforms of fibroblast growth factor (FGF), including aFGF, bFGF, and hst/kFGF. FGF induces ventral, but not the most dorsal, levels of mesodermal tissue; bFGF and its mRNA, and an FGF receptor and its mRNA, are present in the embryo. Thus, FGF probably has a role in mesoderm induction, but is unlikely to be the sole inducing agent in vivo. Second, members of the transforming growth factor-beta (TGF-beta) family. TGF-beta 2 and TGF-beta 3 are active in induction, but the most powerful inducing factors are the distant relatives of TGF-beta named activin A and activin B, which are capable of inducing all types of mesoderm. An important question relates to the establishment of polarity during the induction of mesoderm. While all regions of the animal hemisphere of frog embryos are competent to respond to activins by mesoderm differentiation, only explants that include cells close to the equator form structures with some organization along dorsoventral and anteroposterior axes. These observations suggest that cells in the blastula animal hemisphere are already polarized to some extent, although inducers are required to make this polarity explicit.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity.

Previous experiments have shown that mRNA translation in frog oocytes can be inhibited by the injection of a complementary antisense RNA. Here we explore the use of antisense RNAs to study the functions of localized maternal mRNAs during postfertilization development. While developmental abnormalities were observed in injected fertilized eggs, these abnormalities could not be attributed to the antisense RNA since they were induced at a similar frequency in control embryos. Biochemical tests show that the injected antisense RNA does not form stable hybrids in vivo with its complementary endogenous mRNA. In addition, a novel activity that unwinds RNA:RNA duplexes was found. This activity exists at high levels in eggs and early embryos and is absent or very much diminished in oocytes and late blastula embryos. These results suggest that antisense RNAs may be of limited use in studying the functions of maternal RNAs in Xenopus.

Identification and cloning of localized maternal RNAs from Xenopus eggs.

A central question in developmental biology is to explain how cells in different regions of an embryo acquire different developmental fates. We have begun to address this question by investigating whether specific RNAs are localized within a frog egg. Differential screening of a cDNA library shows that most maternal RNAs are uniformly distributed along the animal-vegetal axis. However, we find that a rare class of maternal RNAs is localized. cDNA clones of four localized RNAs have been characterized. Three of these cDNAs are derived from maternal RNAs that are concentrated in the animal hemisphere of unfertilized eggs and remain localized through the early blastula stage. One cDNA is derived from a maternal RNA found almost exclusively in the vegetal hemisphere at both stages. These studies show that some informational molecules, specifically RNAs, are localized in eggs and are inherited by particular blastomeres.

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).