The one-eyed pinhead gene functions in mesoderm and endoderm formation in zebrafish and interacts with no tail

The zebrafish locus one-eyed pinhead (oep) is essential for the formation of anterior axial mesoderm, endoderm and ventral neuroectoderm. At the beginning of gastrulation anterior axial mesoderm cells form the prechordal plate and express goosecoid (gsc) in wild-type embryos. In oep mutants the prechordal plate does not form and gsc expression is not maintained. Exposure to lithium, a dorsalizing agent, leads to the ectopic induction and maintenance of gsc expression in wild-type embryos. Lithium treatment of oep mutants still leads to ectopic gsc induction but not maintenance, suggesting that oep acts downstream of inducers of dorsal mesoderm. In genetic mosaics, wild-type cells are capable of forming anterior axial mesoderm in oep embryos, suggesting that oep is required in prospective anterior axial mesoderm cells before gastrulation. The oep gene is also essential for endoderm formation and the early development of ventral neuroectoderm, including the floor plate. The loss of endoderm is already manifest during gastrulation by the absence of axial-expressing cells in the hypoblast of oep mutants. These findings suggest that oep is also required in lateral and ventral regions of the gastrula margin. The sonic hedgehog (shh).gene is expressed in the notochord of oep animals. Therefore, the impaired floor plate development in oep mutants is not caused by the absence of the floor plate inducer shh. This suggests that oep is required downstream or in parallel to shh signaling. The ventral region of the forebrain is also absent in oep mutants, leading to severe cyclopia. In contrast, anterior-posterior brain patterning appears largely unaffected, suggesting that underlying prechordal plate is not required for anterior-posterior pattern formation but might be involved in dorsoventral brain patterning. To test if oep has a wider, partially redundant role, we constructed double mutants with two other zebrafish loci essential for patterning during gastrulation. Double mutants with floating head, the zebrafish Xnot homologue, display enhanced floor plate and adaxial muscle phenotypes. Double mutants with no tail (ntl), the zebrafish homologue of the mouse Brachyury locus, display severe defects in midline and mesoderm formation including absence of most of the somitic mesoderm. These results reveal a redundant function of oep and ntl in mesoderm formation. Our data suggest that both oep and ntl act in the blastoderm margin to specify mesendodermal cell fates.     

A role for Siamois in Spemann organizer formation

The vertebrate body plan is specified in the early embryo through the inductive influence of the organizer, a special region that forms on the dorsalmost side of the embryo at the beginning of gastrulation. In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in the area of organizer activity and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, we made a dominant repressor construct (SE) in which the Siamois homeodomain was fused to an active repression domain of Drosophila engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not ventrolateral, marginal zone markers. At similar expression levels, SE proteins with altered DNA-binding specificity do not have the same effect. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. Furthermore, SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development.     

Determination of the zebrafish forebrain: induction and patterning

We report an analysis of forebrain determination and patterning in the zebrafish Danio rerio. In order to study these events, we isolated zebrafish homologs of two neural markers, odd-paired-like (opl), which encodes a zinc finger protein, and fkh5, which encodes a forkhead domain protein. At mid-gastrula, expression of these genes defines a very early pattern in the presumptive neurectoderm, with opl later expressed in the telencephalon, and fkh5 in the diencephalon and more posterior neurectoderm. Using in vitro explant assays, we show that forebrain induction has occurred even earlier, by the onset of gastrulation (shield stage). Signaling from the early gastrula shield, previously shown to be an organizing center, is sufficient for activation of opl expression in vitro. In order to determine whether the organizer is required for opl regulation, we removed from late blastula stage embryos either the presumptive prechordal plate, marked by goosecoid (gsc) expression, or the entire organizer, marked by chordin (chd) expression. opl was correctly expressed after removal of the presumptive prechordal plate and consistently, opl was correctly expressed in one-eyed pinhead (oep) mutant embryos, where the prechordal plate fails to form. However, after removal of the entire organizer, no opl expression was observed, indicating that this region is crucial for forebrain induction. We further show that continued organizer function is required for forebrain induction, since beads of BMP4, which promotes ventral fates, also prevented opl expression when implanted during gastrulation. Our data show that forebrain specification begins early during gastrulation, and that a wide area of dorsal mesendoderm is required for its patterning.     

Zebrafish organizer development and germ-layer formation require nodal-related signals

The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-beta (TGF-beta) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-beta superfamily that is related to mouse nodal. cyc encodes another nodal-related proteins, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.     

Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16

The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.     

The Xcad-2 gene can provide a ventral signal independent of BMP-4

Patterning of the marginal zone in the Xenopus embryo has been attributed to interactions between dorsal genes expressed in the organizer and ventral-specific genes. In this antagonistic interplay of activities, BMP-4, a gene that is not expressed in the organizer, provides a strong ventralizing signal. The Xenopus caudal type homeobox gene, Xcad-2, which is expressed around the blastopore with a gap over the dorsal lip, was analyzed as part of the ventral signal. Xcad-2 was shown to efficiently repress during early gastrula stages the dorsal genes gsc, Xnot-2, Otx-2, XFKH1 and Xlim-1, while it positively regulates the ventral genes, Xvent-1 and Xvent-2, with Xpo exhibiting a strong positive response to Xcad-2 overexpression. Xcad-2 was also capable of inducing BMP-4 expression in the organizer region. Support for a ventralizing role for Xcad-2 was obtained from co-injection experiments with the dominant negative BMP receptor which was used to block BMP-4 signaling. Under lack-of-BMP-signaling conditions Xcad-2 could still regulate dorsal and ventral gene expression and restore normal development, suggesting that it can act downstream of BMP-4 signaling or independently of it. Xcad-2 could also inhibit secondary axis formation and dorsalization induced by the dominant negative BMP receptor. Xcad-2 was also shown to efficiently reverse the dorsalizing effects of LiCl. These results place Xcad-2 as part of the ventralizing gene program which acts during early gastrula stages and can execute its ventralizing function in the absence of BMP signaling.     

Attenuation of kindling-induced decreases in NT-3 mRNA by thyroid hormone depletion

Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a 'loss of function' mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.