Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.     

Alternative splicing results in RET isoforms with distinct trafficking properties

RET encodes a receptor tyrosine kinase that is essential for spermatogenesis, development of the sensory, sympathetic, parasympathetic, and enteric nervous systems and the kidneys, as well as for maintenance of adult midbrain dopaminergic neurons. RET is alternatively spliced to encode multiple isoforms that differ in their C-terminal amino acids. The RET9 and RET51 isoforms display unique levels of autophosphorylation and have differential interactions with adaptor proteins. They induce distinct gene expression patterns, promote different levels of cell differentiation and transformation, and play unique roles in development. Here we present a comprehensive study of the subcellular localization and trafficking of RET isoforms. We show that immature RET9 accumulates intracellularly in the Golgi, whereas RET51 is efficiently matured and present in relatively higher amounts on the plasma membrane. RET51 is internalized faster after ligand binding and undergoes recycling back to the plasma membrane. This differential trafficking of RET isoforms produces a more rapid and longer duration of signaling through the extracellular-signal regulated kinase/mitogen-activated protein kinase pathway downstream of RET51 relative to RET9. Together these differences in trafficking properties contribute to some of the functional differences previously observed between RET9 and RET51 and establish the important role of intracellular trafficking in modulating and maintaining RET signaling.     

Expression of GFRalpha1 receptor splicing variants with different biochemical properties is modulated during kidney development

The glial cell line-derived neurotrophic factor (GDNF) family coreceptor alpha1 (GFRalpha1) is a critical component of the RET receptor kinase signal-transducing complex. The activity of this multicomponent receptor is stimulated by the glial cell line-derived neurotrophic factor (GDNF) and is involved in neuronal cells survival and kidney development. GFRalpha1 pre-mRNA is alternatively spliced and produces two isoforms: GFRalpha1a, which includes the exon 5; and GFRalpha1b, which excludes it. Here we show that the Gfralpha1a isoform is predominantly expressed in neuronal tissues and in PC12 cells differentiated toward a neuronal phenotype. GFRalpha1 splicing is also regulated during kidney development, GFRalpha1a is the minor isoform before birth and then rapidly becomes the major form after birth. We established cell lines expressing either GFRalpha1 isoforms and demonstrated that the GFRalpha1b isoform binds GDNF more efficiently than GFRalpha1a. Consistently, GFRalpha1b promotes a stronger RET phosphorylation than GFRalpha1a. These results indicate that specific inclusion of the GFRalpha1 exon 5 in neuronal tissues or during kidney development may alter the binding properties of GDNF to GFRalpha1, and thus could constitute an additional regulatory mechanism of the RET signaling pathway.     

BMP signalling in early Xenopus development

Bone morphogenetic proteins (BMPs) are typically members of the transforming growth factor β (TGF-β) family with diverse roles in embryonic development. At least five genes with homology to BMPs are expressed during Xenopus development, along with their receptors and intracellular signalling pathways. The evidence suggests that BMPs have roles to play in both mesoderm induction and dorsoventral patterning. Studies in Xenopus have also identified a number of inhibitory binding proteins for the classical BMPs, encoded by genes such as chordin and noggin. These proteins appear to be responsible for establishing a morphogen gradient of BMP4 activity, which specifies different dorsoventral fates in early gastrulae. An emerging theme is that inhibition of BMP signalling is an important mechanism regulating cell fate decisions in early development. BioEssays 21:751–760, 1999. © 1999 John Wiley & Sons, Inc.     

Sexually distinct development of vocal pathways in Xenopus laevis

Deterministic rules, rather than experience, are thought to regulate the development of simple behaviors in vertebrates and invertebrates. We revisited this issue through examination of the sexually distinct vocalizations of African clawed frogs (Xenopus laevis), a reproductive behavior used by sexually mature males and females. We discovered that, as expected for simple behavior, female vocalizations develop through deterministic rules. The rare calls of juvenile females are indistinguishable from those of adult females. The vocal pathways of juvenile females, as measured by the contractile properties of the laryngeal muscles (the vocal muscles) and the laryngeal motoneuron somata (vocal motoneurons) size, are the developmental default and do not differentiate as they mature. Male Xenopus, in contrast, produce extensive vocalizations with rudimentary acoustic structure before reaching sexual maturity. Moreover, the functional properties of the vocal central pattern generator mature before muscle fibers and motoneuron size are fully masculinized. The results suggest that neuronal activity during development may be important in organizing the contractile properties of the muscle fibers in male, but not in female Xenopus. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 862–874, 2010     

H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration

In many systems, ion flows and long-term endogenous voltage gradients regulate patterning events, but molecular details remain mysterious. To establish a mechanistic link between biophysical events and regeneration, we investigated the role of ion transport during Xenopus tail regeneration. We show that activity of the V-ATPase H(+) pump is required for regeneration but not wound healing or tail development. The V-ATPase is specifically upregulated in existing wound cells by 6 hours post-amputation. Pharmacological or molecular genetic loss of V-ATPase function and the consequent strong depolarization abrogates regeneration without inducing apoptosis. Uncut tails are normally mostly polarized, with discrete populations of depolarized cells throughout. After amputation, the normal regeneration bud is depolarized, but by 24 hours post-amputation becomes rapidly repolarized by the activity of the V-ATPase, and an island of depolarized cells appears just anterior to the regeneration bud. Tail buds in a non-regenerative ;refractory' state instead remain highly depolarized relative to uncut or regenerating tails. Depolarization caused by V-ATPase loss-of-function results in a drastic reduction of cell proliferation in the bud, a profound mispatterning of neural components, and a failure to regenerate. Crucially, induction of H(+) flux is sufficient to rescue axonal patterning and tail outgrowth in otherwise non-regenerative conditions. These data provide the first detailed mechanistic synthesis of bioelectrical, molecular and cell-biological events underlying the regeneration of a complex vertebrate structure that includes spinal cord, and suggest a model of the biophysical and molecular steps underlying tail regeneration. Control of H(+) flows represents a very important new modality that, together with traditional biochemical approaches, may eventually allow augmentation of regeneration for therapeutic applications.     

TGF-beta signalling pathways in early Xenopus development

Many different ligands of the TGF-beta superfamily signal in the early Xenopus embryo and are required for the specification and patterning of the three germ layers as well as for gastrulation. Recent advances in the field are helping us understand how ligand activity is regulated both spatially and temporally, the mechanism by which the signals are transduced to the nucleus and how essentially the same signalling pathway can activate completely different sets of genes in different regions of the embryo.     

Xenopus hoxc8 during early development

Vertebrate hoxc8 homologous genes have been shown to be involved in the formation of lower thoracic/lumbar vertebrae during early embryonic development. We report the isolation of a Xenopus hoxc8 (Xhoxc8), which shows 94% amino acid sequence identity to the mouse counterpart. Xhoxc8 is initially expressed in a broad region of blastopore lip at gastrular stage; however, at later stages, the region of expression is progressively restricted to the dorsal region caudal to the third somite and to the central trunk region of abdomen. Retinoic acid treatment that caused a severe malformation in antero-posterior axis did not induce any significant change in the spatio-temporal expression pattern of Xhoxc8 mRNA. Antisense RNA injection into 2- or 4-cell stage embryos resulted in a severe malformation in the abdominal structure leading to embryonic death. The results strongly indicate that Xhoxc8 expression is critical for the formation of abdominal structure.     

Two structural variants of Nek2 kinase, termed Nek2A and Nek2B, are differentially expressed in Xenopus tissues and development

Nek2 kinase, a NIMA-related kinase, has been suggested to play both meiotic and mitotic roles in mammals, but its function(s) during development is poorly understood. We have isolated here cDNAs encoding a Xenopus homolog of mammalian Nek2 and have shown that Xenopus Nek2 has two structural variants, termed Nek2A and Nek2B. Nek2A, most likely a C-terminally spliced form, corresponds to the previously described human and mouse Nek2, while Nek2B is most probably a novel, C-terminally unspliced form of Nek2. As a consequence of this (probable) alternative splicing, Nek2B lacks the C-terminal 70-amino-acid sequence of Nek2A, which contains a PEST sequence (or a motif for rapid degradation). Western blot analysis reveals that Nek2A is expressed predominantly in the testis (presumably in spermatocytes) and very weakly in the stomach and, during development, only after the neurula stage. By contrast, Nek2B is expressed mainly in the ovary and in both primary and secondary oocytes and early embryos up to the neurula stage. These results suggest that Nek2A and Nek2B may play both meiotic and mitotic roles, but in a spatially and temporally complementary manner during Xenopus development, and that Nek2B, rather than Nek2A (or the conventional form of Nek2), may play an important role in early development. We discuss the possibility that a counterpart of Xenopus Nek2B might also exist and function in early mammalian development.