Eukaryotic initiation factor 6 (eif6) overexpression affects eye development in Xenopus laevis

The translation initiation factor eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 serine 235 by PKC. Previous data showed that eif6 binds to the 60S ribosomal subunit when unphosphorylated, inhibiting assembly with the 40S subunit. Phosphorylation of Ser235 releases eif6 from the 60S subunit and allows assembly. eif6 acts as an anti-apoptotic factor via regulation of the bcl2/bax balance and acts selectively upstream of bcl2. This activity also depends upon phosphorylation of eif6 Ser235. One of the consequences of eif6 overexpression in Xenopus embryos is aberrant eye development. Here we evaluate the eye phenotype and show that it is transient. We show that the whole eye, particularly the retina layers, of the embryos injected with eif6-encoding mRNA recover by stage 42. Embryos over-expressing eif6 have normal expression of anterior- and brain-specific markers, indicating that outside the eye field, other neural regions appear unaffected by the eif6 injection. No eye defect was detected when morpholinos were used to reduce eif6 protein synthesis. We tested how two known pathways of eif6 function with respect to alteration of eye development. We found that injection of bcl2 did not produce the eye phenotype and eif6-bax co-injection did not rescue the eye defect, suggesting that the eye phenotype is not bearing on the anti-apoptotic role played by eif6 is not linked to its role as an anti-apoptotic factor. We also determined that PKC-dependant phosphorylation of Ser235 in eif6 is not required to produce defective eye development. These results indicate that the aberrant eye phenotype, produced by eif6 overexpression, is not directly linked to the PKC-regulated effects of eif6 on translation and ribosomal subunit interaction or on eif6 anti-apoptotic properties.     

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.     

Differential perturbations in the morphogenesis of anterior structures induced by overexpression of truncated XB- and N-cadherins in Xenopus embryos

Cadherins, a family of Ca-dependent adhesion molecules, have been proposed to act as regulators of morphogenetic processes and to be major effectors in the maintenance of tissue integrity. In this study, we have compared the effects of the expression of two truncated cadherins during early neurogenesis in Xenopus laevis. mRNA encoding deleted forms of XB- and N-cadherin lacking most of the extracellular domain were injected into the four animal dorsal blastomeres of 32-cell stage Xenopus embryos. These truncated cadherins altered the cohesion of cells derived from the injected blastomeres and induced morphogenetic defects in the anterior neural tissue to which they chiefly contributed. Truncated XB-cadherin was more efficient than N- cadherin in inducing these perturbations. Moreover, the coexpression of both truncated cadherins had additive perturbation effects on neural development. The two truncated cadherins can interact with the three known catenins, but with distinct affinities. These results suggest that the adhesive signal mediated by cadherins can be perturbed by overexpressing their cytoplasmic domains by competing with different affinity with catenins and/or a common anchor structure. Therefore, the correct regulation of cadherin function through the cytoplasmic domain appears to be a crucial step in the formation of the neural tissue.     

Effects of light and dark upon photoreceptor synapses in the retina of Xenopus laevis

Summary The adrenergic innervation of the juxtaglomerular complex was studied in kidneys from mice, rats, guinea-pigs, rabbits, cats, dogs, pigs, monkeys, and humans using fluorescence histochemistry of neuronal nor-adrenaline and autoradiography of ³H-noradrenaline. The localization of the nerves was established by phase contrast optics or by perfusing the vascular system with India ink. Adrenergic nerve terminals, exhibiting a formaldehyde-induced fluorescence and having the ability to take up and accumulate ³H-noradrenaline, were easily identified when they enclosed the glomerular afferent arteriole. They continued in between and close to the macula densa and lacis cells to supply the glomerular efferent arteriole. The nerves could be seen to accompany this arteriole for a considerable distance until they branched off to the vasa recta in the juxtamedullary region and to adjacent cortical veins. This innervation pattern was found to be a constant feature except in kidneys from guinea-pigs and cats, in which post-glomerular adrenergic nerves were not found in some of the superficial glomerular units. The fluorescence in all adrenergic fibres supplying the juxtaglomerular complex disappeared after removal of the aortico-renal ganglion, showing that they belong to a common system of renal sympathetic nerves.This work is dedicated to Professor Wolfgang Bargmann in honour of his seventieth birthday, January 26, 1976     

Desensitization of IP3-induced Ca2+ release by overexpression of a constitutively active Gqalpha protein converts ventral to dorsal fate in Xenopus early embryos

The constitutively active Gqalpha mutant construct (GqalphaQ-L) in Xenopus early embryos was overexpressed and the effects on dorsoventral patterning examined. It was found that prolonged stimulation of inositol 1,4,5-trisphosphate (IP3)-Ca2+ signaling by overexpression of GqalphaQ-L led to desensitization of IP3-induced Ca2+ release (IICR). Desensitization of IICR on the ventral side specifically induced an ectopic dorsal axis due to the conversion of ventral marginal mesoderm to adopt a dorsal fate. This effect of desensitization resembles that of inhibitory antibodies against the IP3 receptor, as reported previously. These results strengthen the earlier finding that active IP3-Ca2+ signaling functions in ventral signaling during the early embryonic development of Xenopus. Furthermore, the nature of downregulation of the Xenopus IP3 receptor through continuous stimulation of IP3-Ca2+ signaling might play a role in regulating endogenous IP3-Ca2+ signaling in Xenopus early development.     

Adrenoreceptive structures in the early embryos of the clawed toad (Xenopus laevis)

Studies have been made on the binding of labeled ligands to beta-adrenoreceptor--3H-dihydroalprenolol (DHA) and 125I-iodocyanopindolol (ICP--in crude homogenate and subcellular fractions of toad embryos during cleavage divisions. Specific binding of these ligands was observed which is confirmed by a decrease of the activity after addition of cold propranolol or adrenalin. The value of Kd for DHA is equal to 3 x 10(-9) M for ICP--to 1.5 x 10(-9) M. In addition to this high-affinity pool of binding with a low capacity, a low-affinity and high-capacity pool was also found. Cooling of the preparation up to 4 degrees C for 2 h increases the specific binding simultaneously with a decrease of the affinity of adrenoreceptive structures of the embryo. The data obtained are discussed in relation to the criteria of intracellular transmitter process.     

The multiple decisions made by growth cones of RGCs as they navigate from the retina to the tectum in Xenopus embryos

Retinal ganglion cells (RGCs) of Xenopus laevis send axons along a stereospecific pathway from the retina to their target the optic tectum. Viewed from the point of the growth cone, this journey is reflected by discrete processes of axon initiation, axon outgrowth, navigation, target recognition, and innervation. These processes are characterised by distinct signalling mechanisms that trigger dynamic changes in growth cone morphology and behavior. Here we review work primarily from our laboratory, examining these events from a cellular and molecular perspective, focusing on the roles of FGFs, netrins, receptors, and intracellular effectors.     

Spatio-temporal distribution of the protein of Xenopus vasa homologue (Xenopus vasa-like gene 1, XVLG1) in embryos

In order to know when the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila , the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10–42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.     

Suppressor analysis of fimbrin (Sac6p) overexpression in yeast

Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other.     

Expression of a novel cadherin (EP-cadherin) in unfertilized eggs and early Xenopus embryos

Two distinct cadherin cDNA clones of Xenopus laevis were isolated from a stage 17 embryo cDNA library. Analysis of the complete deduced amino acid sequences indicated that one of these molecules is closely homologous to chicken and mouse N-cadherin, while the other displays comparable homology to both E- and P-cadherins and was thus denoted EP-cadherin. This molecule has an apparent relative molecular mass of 125 x 10(3) (compared to approx. 138 x 10(3) or approx. 140 x 10(3) of E-cadherin and N-cadherins, respectively). Northern and Western blot analyses indicated that N-cadherin is first expressed at the neurula stage while EP-cadherin is the only cadherin detected in unfertilized eggs and cleavage stage embryos. Immunolabeling of Xenopus eggs with antibodies prepared against a fusion protein, containing a segment of EP-cadherin, indicated that the protein is highly enriched at the periphery of the animal hemisphere. EP-cadherin was also found in A6 epithelial cells derived from Xenopus kidneys, and was apparently localized in the intercellular adherens junctions.     

Expression of regulatory genes Px6, Otx2, Six3, and FGF2 during newt retina regeneration

Molecular-genetic mechanisms of regeneration of adult newt (Pleurodeles waltl) retina were studied. For the first time, a comparative analysis of the expression of regulatory genes Pax6, Otx2, and Six3 and FGF2 genes encoding signal molecules was performed in the normal retinal pigment epithelium (RPE) and retina and at successive stages of retina regeneration. Cell differentiation types were determined using genetic markers of cell differentiation in the RPE (RPE65) and the retina (βII-tubulin and Rho). Activation of the expression of neurospecific genes Pax6 and Six3 and the growth factor gene FGF2 and suppression of activation of the regulatory gene Otx2 and the RPE65 were observed at the stage of multipotent neuroblast formation in the regenerating retina. The expression of genes Pax6, Six3, and Fgf2 was retained at a later stage of retina regeneration at which the expression of retinal differentiation markers, the genes encoding β II-tubulin (βII-tubulin) and rhodopsin (Rho), was also detected. We assume that the above regulatory genes are multifunctional and control not only transdifferentiation of RPE cells (the key stage of retina regeneration) but also differentiation of regenerating retina cells. The results of this study, demonstrating coexpression of Pax6, Six3, Fgf2, βII-tubulin, and Rho genes, provide indirect evidence for the interaction of regulatory and specific genes during retina regeneration.     

Rapid deadenylation and Poly(A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3' untranslated region in Xenopus embryos

The 3' untranslated region (3'UTR) of many eukaryotic mRNAs is essential for their control during early development. Negative translational control elements in 3'UTRs regulate pattern formation, cell fate, and sex determination in a variety of organisms. tra-2 mRNA in Caenorhabditis elegans is required for female development but must be repressed to permit spermatogenesis in hermaphrodites. Translational repression of tra-2 mRNA in C. elegans is mediated by tandemly repeated elements in its 3'UTR; these elements are called TGEs (for tra-2 and GLI element). To examine the mechanism of TGE-mediated repression, we first demonstrate that TGE-mediated translational repression occurs in Xenopus embryos and that Xenopus egg extracts contain a TGE-specific binding factor. Translational repression by the TGEs requires that the mRNA possess a poly(A) tail. We show that in C. elegans, the poly(A) tail of wild-type tra-2 mRNA is shorter than that of a mutant mRNA lacking the TGEs. To determine whether TGEs regulate poly(A) length directly, synthetic tra-2 3'UTRs with and without the TGEs were injected into Xenopus embryos. We find that TGEs accelerate the rate of deadenylation and permit the last 15 adenosines to be removed from the RNA, resulting in the accumulation of fully deadenylated molecules. We conclude that TGE-mediated translational repression involves either interference with poly(A)'s function in translation and/or regulated deadenylation.