ZFPIP/Zfp462 is maternally required for proper early Xenopus laevis development

ZFPIP (Zinc Finger Pbx1 Interacting Protein) has been recently identified in our laboratory in a yeast two hybrid screen using an embryonic mouse cDNA library and PBX1 as a bait. This gene encodes a large protein (250 kDa) that contains a bipartite NLS, numerous C2H2 zinc fingers and is highly conserved amongst vertebrates. In order to address the role of ZFPIP during embryonic development, we analysed the expression pattern of the gene and performed morpholinos injections into Xenopus laevis embryos. We first showed that the ZFPIP protein was maternally present in oocytes. Then, ZFPIP was detected from morula to neurula stages in the nucleus of the cells, with a gradient from animal to vegetal pole. By injection of ZFPIP morpholinos, we showed that morphant embryos were unable to undergo proper gastrulation and subsequently exhibited a persistent opened blastopore. Analysis of molecular and cellular events that were altered in morphant embryos highlighted an impairment of cell division processes as illustrated by atypical mitosis with aberrant metaphase, anaphase or telophase, incomplete chromosome segregation or conjointed nuclei. The overall data presented here demonstrated that ZFPIP was a major developing gene that acts in the very first steps of embryonic development of Xenopuslaevis.     

Thyroid hormone receptor subtype specificity for hormone-dependent neurogenesis in Xenopus laevis

Thyroid hormone (T₃) influences cell proliferation, death and differentiation during development of the central nervous system (CNS). Hormone action is mediated by T₃ receptors (TR) of which there are two subtypes, TRα and TRβ. Specific roles for TR subtypes in CNS development are poorly understood. We analyzed involvement of TRα and TRβ in neural cell proliferation during metamorphosis of Xenopus laevis. Cell proliferation in the ventricular/subventricular neurogenic zones of the tadpole brain increased dramatically during metamorphosis. This increase was dependent on T₃ until mid-prometamorphosis, after which cell proliferation decreased and became refractory to T₃. Using double labeling fluorescent histochemistry with confocal microscopy we found TRα expressed throughout the tadpole brain, with strongest expression in proliferating cells. By contrast, TRβ was expressed predominantly outside of neurogenic zones. To corroborate the histochemical results we transfected living tadpole brain with a Xenopus TRβ promoter-EGFP plasmid and found that most EGFP expressing cells were not dividing. Lastly, treatment with the TRα selective agonist CO23 increased brain cell proliferation; whereas, treatment with the TRβ-selective agonists GC1 or GC24 did not. Our findings support the view that T₃ acts to induce cell proliferation in the tadpole brain predominantly, if not exclusively, via TRα.     

XsFRP5 modulates endodermal organogenesis in Xenopus laevis

Canonical Wnt signalling is known to be involved in the regulation of differentiation and proliferation in the context of endodermal organogenesis. Wnt mediated β-catenin activation is understood to be modulated by secreted Frizzled-related proteins, such as XsFRP5, which is dynamically expressed in the prospective liver/ventral pancreatic precursor cells during late neurula stages, becoming liver specific at tailbud stages and shifting to the posterior stomach/anterior duodenum territory during tadpole stages of Xenopus embryogenesis. These expression characteristics prompted us to analyse the function of XsFRP5 in the context of endodermal organogenesis. We demonstrate that XsFRP5 can form a complex with and inhibit a multitude of different Wnt ligands, including both canonical and non-canonical ones. Knockdown of XsFRP5 results in transient pancreatic hypoplasia as well as in an enlargement of the stomach. In VegT-injected animal cap explants, XsFRP5 can induce expression of exocrine but not endocrine pancreatic marker genes. Both, its expression characteristics as well as its interactions with XsFRP5, define Wnt2b as a putative target for XsFRP5 in vivo. Knockdown of Wnt2b results in a hypoplastic stomach as well as in hypoplasia of the pancreas. On the basis of these findings we propose that XsFRP5 exerts an early regulatory function in the specification of the ventral pancreas, as well as a late function in controlling stomach size via inhibition of Wnt signalling.     

Identification and preliminary functional analysis of alternative splicing of Siah1 in Xenopus laevis

Siah proteins are vertebrate homologs of the Drosophila ‘seven in absentia’ gene. In this study, we characterized two splicing forms, Siah1a and Siah1b, of the Xenopus seven in absentia homolog 1 gene (Siah1). Overexpression of xSiah1a led to severe suppression of embryo cleavage, while that of xSiah1b was not effective even at a high dose. Competition analysis demonstrated that co-expression of xSiah1a and 1b generated the same phenotype as overexpression of xSiah1a alone, suggesting that xSiah1b does not interfere with the function of xSiah1a. Since xSiah1b has an additional 31 amino acids in the N-terminus compared to xSiah1a, progressive truncation of xSiah1b from the N-terminus showed that inability of xSiah1b to affect embryo cleavage was associated with the length of the N-terminal extension of extra amino acids. The possible implication of this finding is discussed.     

Caerulein precursor fragment (CPF) peptides from the skin secretions of Xenopus laevis and Silurana epitropicalis are potent insulin-releasing agents

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid clawed frog Xenopus laevis (Pipidae) led to the identification of 10 peptides with the ability to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line. These peptides were purified to near homogeneity and structural characterization showed that they belong to the magainin (2 peptides), peptide glycine-leucine-amide (PGLa) (1 peptide), xenopsin precursor fragment (1 peptide), and caerulein precursor fragment (CPF) (6 peptides) families. CPF-1, CPF-3, CPF-5 and CPF-6 were the most potent producing a significant (P < 0.05) increase in the rate of insulin release at concentration of 0.03 nM. CPF-7 (GFGSFLGKALKAALKIGANALGGAPQQ) produced the maximum stimulation of insulin release (571 ± 30% of basal rate at 3 μM). In addition, CPF-SE1 (GFLGPLLKLGLKGVAKVIPHLIPSRQQ), previously isolated from skin secretions of the tetraploid frog Silurana epitropicalis, produced a significant (P < 0.05) increase in the rate of insulin release at 0.03 nM with a 514 ± 13% increase over basal rate at 3 μM. No CPF peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from BRIN-BD11 cells at concentrations up to 3 μM indicating that the integrity of the plasma membrane had been preserved. The mechanism of action of the CPF peptides involves, at least in part, membrane depolarization and an increase in intracellular Ca²⁺ concentration. The CPF peptides show potential for development into agents for the treatment of Type 2 diabetes.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Delta-Notch signaling is involved in the segregation of the three germ layers in Xenopus laevis

In vertebrates, the induction of the three germ layers (ectoderm, mesoderm and endoderm) has been extensively studied, but less is known about how they segregate. Here, we investigated whether Delta-Notch signaling is involved in this process. Activating the pathway in the marginal zone with Notch^ICD resulted in an expansion of endodermal and neural ectoderm precursors, leaving a thinner mesodermal ring around the blastopore at gastrula stage, when germ layers are segregated. On the other hand, when the pathway was blocked with Delta-1^STU or with an antisense morpholino oligonucleotide against Notch, the pan-mesodermal brachyury (bra) domain was expanded and the neural border was moved animalwards. Strikingly, the suprablastoporal endoderm was either expanded when Delta-1 signaling was blocked, or reduced after the general knock-down of Notch. In addition, either activating or blocking the pathway delays the blastopore closure. We conclude that the process of delimiting the three germ layers requires Notch signaling, which may be finely regulated by ligands and/or involve non-canonical components of the pathway. Moreover, Notch activity must be modulated at appropriate levels during this process in order to keep normal morphogenetic movements during gastrulation.     

An environmentally relevant endocrine-disrupting antiandrogen, vinclozolin, affects calling behavior of male Xenopus laevis

Vinclozolin (VIN) is an antiandrogenic model substance as well as a common fungicide that can affect the endocrine system of vertebrates. The objective of this study was to investigate how VIN affects mate calling behavior of South African clawed frogs (Xenopus laevis) and whether it is effective at environmentally relevant concentrations. Male X. laevis were injected with human chorionic gonadotropin (hCG) to stimulate their androgen-controlled mate calling behavior and were treated with VIN at concentrations of 10⁻⁶, 10⁻⁸ and 10⁻¹⁰ M. VIN at 10⁻⁶ M reduced calling activity. Furthermore, the vocalization composition of VIN-treated X. laevis was altered. The call types advertisement calls and chirping are uttered by reproductively active males, whereas the call types growling, ticking, and rasping indicate a sexually unaroused state of a male. VIN at any of the tested concentrations led to a decrease in utterance of calls, which indicate a sexually aroused state of the males, and an increase in relative proportions of calls, indicating a sexually unaroused state of the males. Additionally, the mean duration of clicks and the number of accentuated clicks during the advertisement calls decreased at all concentrations of VIN. No significant differences were observed in any other temporal or spectral calling parameters between the treatments. This study illustrates that exposure to the antiandrogen VIN might result in a reduced reproductive success by altering mate calling behavior of X. laevis. Moreover, it suggests that the behavioral parameters examined in this study can be used as sensitive biomarkers for detecting antiandrogenic endocrine disrupting compounds in amphibians.     

Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6

Mechanisms coupling heart function and cardiac morphogenesis can be accessed in lower vertebrate embryos that can survive to swimming tadpole stages on diffused oxygen. Forward genetic screens in Xenopus tropicalis have identified more than 80 mutations affecting diverse developmental processes, including cardiac morphogenesis and function. In the first positional cloning of a mutation in X. tropicalis, we show that non-contractile hearts in muzak (muz) embryos are caused by a premature stop codon in the cardiac myosin heavy chain gene myh6. The mutation deletes the coiled-coil domain responsible for polymerization into thick filaments, severely disrupting the cardiomyocyte cytoskeleton. Despite the lack of contractile activity and absence of a major structural protein, early stages of cardiac morphogenesis including looping and chamber formation are grossly normal. Muz hearts subsequently develop dilated chambers with compressed endocardium and fail to form identifiable cardiac valves and trabeculae.     

Reactive oxygen species and anti-oxidant defenses in tail of tadpoles, Xenopus laevis

Tail regression in tadpoles is one of the most spectacular events in anuran metamorphosis. Reactive oxygen species and oxidative stress play an important role during this process. Presently, the cell- and tissue-specific localization of antioxidant enzymes such as superoxide dismutase (SOD) and catalase as well as neuronal and inducible nitric oxide synthase isoforms (nNOS and iNOS) responsible for production of nitric oxide (NO) were carried out during different stages of metamorphosis in tail of tadpole Xenopus laevis. NO also has profound effect on the mitochondrial function having its own nitric oxide NOS enzyme. Hence, in situ staining for NO and mitochondria also was investigated. The distribution of nNOS and iNOS was found to be stage specific, and the gene expression of nNOS was up-regulated by thyroxin treatment. In situ staining for NO and mitochondria shows co-localization, suggesting mitochondria being one of the sources of NO. SOD and catalase showed significant co-localization during earlier stages of metamorphosis, but before the tail regression begins, there was a significant decrease in activity as well as co-localization suggesting increased ROS accumulation. These findings are discussed in terms of putative functional importance of ROS and cytoplasmic as well as mitochondrial derived NO in programmed cell death in tail tissue.     

Left-right asymmetric morphogenesis of the anterior midgut depends on the activation of a non-muscle myosin II in Drosophila

Many animals exhibit stereotypical left-right (LR) asymmetry in their internal organs. The mechanisms of LR axis formation required for the subsequent LR asymmetric development are well understood, especially in some vertebrates. However, the molecular mechanisms underlying LR asymmetric morphogenesis, particularly how mechanical force is integrated into the LR asymmetric morphogenesis of organs, are poorly understood. Here, we identified zipper (zip), encoding a Drosophila non-muscle myosin II (myosin II) heavy chain, as a gene required for LR asymmetric development of the embryonic anterior midgut (AMG). Myosin II is known to directly generate mechanical force in various types of cells during morphogenesis and cell migration. We found that myosin II was involved in two events in the LR asymmetric development of the AMG. First, it introduced an LR bias to the directional position of circular visceral muscle (CVMU) cells, which externally cover the midgut epithelium. Second, it was required for the LR-biased rotation of the AMG. Our results suggest that myosin II in CVMU cells plays a crucial role in generating the force leading to LR asymmetric morphogenesis. Taken together with previous studies in vertebrates, the involvement of myosin II in LR asymmetric morphogenesis might be conserved evolutionarily.     

The Xenopus Irx genes are essential for neural patterning and define the border between prethalamus and thalamus through mutual antagonism with the anterior repressors Fezf and Arx

The Iroquois (Irx) genes encode homeoproteins conserved during evolution. Vertebrate genomes contain six Irx genes organized in two clusters, IrxA (which harbors Irx1, Irx2 and Irx4) and IrxB (which harbors Irx3, Irx5 and Irx6). To determine the precise role of these genes during development and their putative redundancies, we conducted a comparative expression analysis and a comprehensive loss-of-function study of all the early expressed Irx genes (Irx1-5) using specific morpholinos in Xenopus. We found that the five Irx genes display largely overlapping expression patterns and contribute to neural patterning. All Irx genes are required for proper formation of posterior forebrain, midbrain, hindbrain and, to a lesser an extent, spinal cord. Nevertheless, Irx1 and Irx3 seem to have a predominant role during regionalization of the neural plate. In addition, we find that the common anterior limit of Irx gene expression, which will correspond to the future border between the prethalamus and thalamus, is defined by mutual repression between Fezf and Irx proteins. This mutual repression is likely direct. Finally, we show that Arx, another anteriorly expressed repressor, also contribute to delineate the anterior border of Irx expression.