Development Of Xenopus Laevis Research Articles

Coordinating heart morphogenesis: A novel role for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels during cardiogenesis in Xenopus laevis

ABSTRACTHyperpolarization-activated cyclic-nucleotide gated channel (HCN) proteins are important regulators of both neuronal and cardiac excitability. Among the 4 HCN isoforms, HCN4 is known as a pacemaker channel, because it helps control the periodicity of contractions in vertebrate hearts. Although the physiological role of HCN4 channel has been studied in adult mammalian hearts, an earlier role during embryogenesis has not been clearly established. Here, we probe the embryonic roles of HCN4 channels, providing the first characterization of the expression profile of any of the HCN isoforms during Xenopus laevis development and investigate the consequences of altering HCN4 function on embryonic pattern formation. We demonstrate that both overexpression of HCN4 and injection of dominant-negative HCN4 mRNA during early embryogenesis results in improper expression of key patterning genes and severely malformed hearts. Our results suggest that HCN4 serves to coordinate morphogenetic control factors that provide positional information during heart morphogenesis in Xenopus.

Frizzled 3 acts upstream of Alcam during embryonic eye development

Formation of a functional eye during vertebrate embryogenesis requires different processes such as cell differentiation, cell migration, cell-cell interactions as well as intracellular signalling processes. It was previously shown that the non-canonical Wnt receptor Frizzled 3 (Fzd3) is required for proper eye formation, however, the underlying mechanism is poorly understood. Here we demonstrate that loss of Fzd3 induces severe malformations of the developing eye and that this defect is phenocopied by loss of the activated leukocyte cell adhesion molecule (Alcam). Promoter analysis revealed the presence of a Fzd3 responsive element within the alcam promoter, which is responsible for alcam expression during anterior neural development. In-depth analysis identified the jun N-terminal protein kinase 1 (JNK1) and the transcription factor paired box 2 (Pax2) to be important for the activation of alcam expression. Altogether our study reveals that alcam is activated through non-canonical Wnt signalling during embryonic eye development in Xenopus laevis and shows that this pathway plays a similar role in different tissues.

Pattern of Neurogenesis and Identification of Neuronal Progenitor Subtypes during Pallial Development in Xenopus laevis

The complexity of the pallium during evolution has increased dramatically in many different respects. The highest level of complexity is found in mammals, where most of the pallium (cortex) shows a layered organization and neurons are generated during development following an inside-out order, a sequence not observed in other amniotes (birds and reptiles). Species-differences may be related to major neurogenetic events, from the neural progenitors that divide and produce all pallial cells. In mammals, two main types of precursors have been described, primary precursor cells in the ventricular zone (vz; also called radial glial cells or apical progenitors) and secondary precursor cells (called basal or intermediate progenitors) separated from the ventricle surface. Previous studies suggested that pallial neurogenetic cells, and especially the intermediate progenitors, evolved independently in mammalian and sauropsid lineages. In the present study, we examined pallial neurogenesis in the amphibian Xenopus laevis, a representative species of the only group of tetrapods that are anamniotes. The pattern of pallial proliferation during embryonic and larval development was studied, together with a multiple immunohistochemical analysis of putative progenitor cells. We found that there are two phases of progenitor divisions in the developing pallium that, following the radial unit concept from the ventricle to the mantle, finally result in an outside-in order of mature neurons, what seems to be the primitive condition of vertebrates. Gene expressions of key transcription factors that characterize radial glial cells in the vz were demonstrated in Xenopus. In addition, although mitotic cells were corroborated outside the vz, the expression pattern of markers for intermediate progenitors differed from mammals.

Pax2/Pax8-defined subdomains and the occurrence of apoptosis in the posterior placodal area of mice.

The present work aims to improve our understanding of the causes and functions of apoptosis during the morphogenesis of epibranchial placodes in mice. Schematic maps helped to compare the spatiotemporal sequence of apoptotic events with the protein expression patterns of general (Six1) and specific placodal markers (Pax2, Pax8). Our findings challenge the view that, in mammals, all three epibranchial placodes spring from the original posterior placodal area (PPA) of presomite or early somite embryos. Instead, close-meshed analysis of the Pax2/Pax8 expression patterns demonstrates the stepwise emergence of two subdomains which both belong to the gradually expanding PPA, and which largely give rise to the otic placode and epibranchial placode 1 (anterior subdomain), or to the caudal epibranchial placodes (posterior subdomain). Our observations reinforce previous doubts raised on the PPA progeny of early somite Xenopus embryos (Schlosser and Ahrens, Dev Biol 271:439-466, 2004). They also demonstrate that partly different Pax2/Pax8 codes accompany epibranchial placode development in Xenopus laevis and mice. In mice, interplacodal apoptosis assists in the establishment of the two PPA subdomains and, subsequently, of individualized placodes by predominantly eliminating Six1+ placodal precursor cells. Onset of interplacodal and intraplacodal large-scale apoptosis is almost always preceded and/or paralleled by Pax2/Pax8 expression minima in the very same region. Future work will demand the use of knock-out mice and whole embryo culture to experimentally test, whether the combined action of differentially expressed Pax2 and Pax8 genes exerts antiapoptotic effects in the mammalian PPA.

The Nedd4 binding protein 3 is required for anterior neural development in Xenopus laevis

The Fezzin family member Nedd4-binding protein 3 (N4BP3) is known to regulate axonal and dendritic branching. Here, we show that n4bp3 is expressed in the neural tissue of the early Xenopus laevis embryo including the eye, the brain and neural crest cells. Knockdown of N4bp3 in the Xenopus anterior neural tissue results in severe developmental impairment of the eye, the brain and neural crest derived cranial cartilage structures. Moreover, we demonstrate that N4bp3 depletion leads to a significant reduction of both eye and brain specific marker genes and reduced neural crest cell migration. Finally, we demonstrate an impact of N4bp3 deficiency on cell apoptosis and proliferation. Our studies indicate that N4bp3 is required for early anterior neural development of vertebrates. This is in line with a study implicating that genetic disruption of N4BP3 in humans might be related to neurodevelopmental disease.

Role of Cdc6 During Oogenesis and Early Embryo Development in Mouse and Xenopus laevis.

Cdc6 is an important player in cell cycle regulation. It is involved in the regulation of both S-phase and M-phase. Its role during oogenesis is crucial for repression of the S-phase between the first and the second meiotic M-phases, and it also regulates, via CDK1 inhibition, the M-phase entry and exit. This is of special importance for the reactivation of the major M-phase-regulating kinase CDK1 (Cyclin-Dependent Kinase 1) in oocytes entering metaphase II of meiosis and in embryo cleavage divisions, in which precise timing allows coordination between cell cycle events and developmental program of the embryo. In this chapter, we discuss the role of Cdc6 protein in oocytes and early embryos.

Asymmetric Localization and Distribution of Factors Determining Cell Fate During Early Development of Xenopus laevis.

Asymmetric division is a property of eukaryotic cells that is fundamental to the formation of higher life forms. Despite its importance, the mechanism behind it remains elusive. Asymmetry in the cell is induced by polarization of cell fate determinants that become unevenly distributed among progeny cells. So far dozens of determinants have been identified. Xenopus laevis is an ideal system to study asymmetric cell division during early development, because of the huge size of its oocytes and early-stage blastomeres. Here, we present the current knowledge about localization and distribution of cell fate determinants along the three body axes: animal-vegetal, dorsal-ventral, and left-right. Uneven distribution of cell fate determinants during early development specifies the formation of the embryonic body plan.

FoxD1 protein interacts with Wnt and BMP signaling to differentially pattern mesoderm and neural tissue.

The foxd1 gene (previously known as Brain Factor 2/BF2) is expressed during early Xenopus laevis development. At gastrula stages, foxd1 is expressed in dorsal mesoderm regions fated for muscle and notochord, while at neurula stages, foxd1 is expressed in the forebrain region. Previous studies in the neural plate showed that FoxD1 protein acts as transcriptional repressor downstream of BMP antagonism, neuralizing the embryo to control anterior neural cell fates. FoxD1 mesoderm function was not rigorously analyzed, but ectopic FoxD1 levels increased muscle marker expression in embryos. Using a FoxD1-specific antisense morpholino oligonucleotide, we knocked down endogenous FoxD1 protein activity in developing Xenopus embryos. In this present study, we show that FoxD1 is crucial for dorsal mesoderm formation. Analogous to neural tissue, FoxD1 acts downstream of BMP antagonism to induce dorsal mesoderm cell fates, such as muscle and notochord. FoxD1 is sensitive to its local signaling environment, having differential transcription factor activity in the presence or absence of Wnt or BMP signaling. FoxD1 induces posterior neural tissue in the presence of Wnt or BMP activities, but its activity is restricted to "normal" anterior neural tissue induction when BMP and Wnt activities are repressed. In dorsal mesoderm, FoxD1 interacts with Wnt signaling and BMP antagonism to induce muscle and notochord, while simultaneously repressing more anterior and ventral mesoderm cell fates. FoxD1 protein has multiple activities that are masked or released in the different germ layers as a function of the local signaling environment.

Retinoid-like compounds produced by phytoplankton affect embryonic development of Xenopus laevis

Teratogenic effects, which were remarkably similar to those induced by retinoic acids, have been seen in wild frogs indicating possible source of retinoids in the environment. Recent studies indicate that some cyanobacterial species can contain teratogenic retinoic acids (RAs) and their analogues. Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis. The study investigated the effects of exudates (extracellular compounds) of two cyanobacteria species with retinoic-like activity and one algae species on embryonic development of amphibians. The retinoid-like activity determined by in vitro reporter gene assay reached 528ng retinoid equivalents (REQ)/L and 1000ng REQ/L in exudates of Cylindrospermopsis raciborskii and Microcystis aeruginosa, respectively, while algal exudates showed no detectable activity. Total mean of retinoid-like copounds into exudate was 35.6ng ATRA/mil.cells for M.aeruginosa and 6.71ng ATRA/mil.cells for C.raciborskii, respectively. Toxicity tests with amphibian embryos up to 96h of development were carried out according to the standard guide for the Frog Embryo Teratogenesis Assay Xenopus. Lowest observed effect concentrations (LOEC) of malformations (2.5–2.6µg/L REQ) were two times lower than LOEC for ATRA (5µg/L). The exudates of both cyanobacteria were indeed provoking diverse teratogenic effects (e.g. tail, gut and eyes deformation) and interference with growth in frogs embryos, while such effects were not observed for the algae. Xenopus embryos were also exposed to all-trans retinoic acid (ATRA) in concentration range (1−40µg/L) equivalent to the REQs detected in cyanobacterial exudates. ATRA (10µg/L) caused similar teratogenic phenotypes at corresponding REQs as cyanobacterial exudates. The study confirms the ability of some species of cyanobacteria to produce retinoids naturally and excrete them directly into the environment at concentrations which might have adverse influence on the development of amphibians.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation.

Experimental approaches to the study of the formation of axial structures during early development of Xenopus laevis

The effect of mechanical extension on the differentiation of axial mesoderm in double explants (sandwiches) of Xenopus laevis embryonic tissues isolated during the early gastrula–late neurula developmen-tal period is studied. In explants at the early gastrula stage, artificial extension orients and stimulates isolated differentiation of the notochord and somites as well as their joint formation. Moreover, extension facilitated the formation of the normal anatomical structure of the notochord and affected expression of Chordin gene. At the late gastrula stage, the effect of artificial extension on joint somite–notochord differentiation was weaker. At the stage of late neurula, somites were sometimes formed in explants lacking a notochord anlage. Thus, at earlier stages, the formation of somites was stimulated by contacts with the notochord and joint development of both structures was mechanical dependent, while at the later stages, somites developed inde-pendently of the notochord. Thus, the role of tissue extension is primarily the establishment of normal mor-phology and expression of Chordin was located in the direction of extension.

CFTR-β-catenin interaction regulates mouse embryonic stem cell differentiation and embryonic development.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated anion channel capable of conducting both Cl- and HCO3-, mutations of which cause cystic fibrosis (CF), a common autosomal recessive disease. Although CF patients are known to have varied degree of developmental problems, the biological role of CFTR in embryonic development remains elusive. Here, we show that CFTR is functionally expressed in mouse ESCs. CFTR-/- mESCs exhibit dramatic defect in mesendoderm differentiation. In addition, CFTR physically interacts with β-catenin, defect of which leads to premature degradation of β-catenin and suppressed activation of β-catenin signaling. Furthermore, knockdown of CFTR retards the early development of Xenopus laevis with impaired mesoderm/endoderm differentiation and β-catenin signaling. Our study reveals a previously undefined role of CFTR in controlling ESC differentiation and early embryonic development via its interaction with β-catenin, and provides novel insights into the understanding of embryonic development.

Dietary exposure to polybrominated diphenyl ether 47 (BDE-47) inhibits development and alters thyroid hormone-related gene expression in the brain of Xenopus laevis tadpoles.

Few studies have investigated the thyroid-disrupting effects of polybrominated diphenyl ethers (PBDEs) across multiple levels of biological organization in anurans, despite their suitability for the screening of thyroid disruptors. Therefore, the present study evaluated the effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on development, thyroid histology and thyroid hormone-related gene expression in Xenopus laevis exposed to 0 (control), 50 (low), 500 (medium) or 5000μg BDE-47/g food (high) for 21days. Only the high dose of BDE-47 hindered growth and development; however, thyroid hormone-associated gene expression was downregulated in the brains of tadpoles regardless of dose. These results show that BDE-47 disrupts thyroid hormone signaling at the molecular and whole-organism levels and suggest that gene expression in the brain is a more sensitive endpoint than metamorphosis. Furthermore, the altered gene expression patterns among BDE-47-exposed tadpoles provide insight into the mechanisms of PBDE-induced thyroid disruption and highlight the potential for PBDEs to act as neurodevelopmental toxicants.

β-Catenin Mutations: Insights into the APC Pathway and the Power of Genetics.

See related article by Sparks et al., [Cancer Res 1998;58:1130–4][1] . Visit the Cancer Research 75th Anniversary [timeline][2]. In the mid 1990s, the human genome project was well underway, but it would still be more than 5 years before the first draft of the human genome was published and more

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

Continued Studies on the Effects of Simazine on the Liver Histological Structure and Metamorphosis in the Developing Xenopus laevis.

This study continued our previous work (Sai et al. in Bull Environ Contam Toxicol 95:157-163, 2015a) by analysing the effects of simazine on the liver histological structure and metamorphosis in the developing Xenopus laevis. Tadpoles (Nieuwkoop-Faber stage 46) were exposed to simazine at 0.1, 1.2, 11.0 and 100.9μg/L for 100days. When tadpoles were exposed to simazine at 11.0 and 100.9µg/L, an increased mortality and damaged liver tissues were observed together with significant inhibition of percent of X. laevis completing metamorphosis on days 80 and 90 and prolonged time of completing metamorphosis. On the other hand, we found that simazine has no significant effects on liver weight and altered hepatosomatic index. Results of this study may be considered to inform risk assessment of the effects of simazine on the development of X. laevis.

Effects of Elevated In Ovo Selenium Exposure on Late Stage Development of Xenopus laevis Tadpoles.

The effects of elevated in ovo selenium (Se) exposure on late stage larval Xenopus laevis development were investigated. Adult females were fed diets augmented with selenomethionine for 68days and bred with untreated males to obtain embryos with measured concentrations of 1.6 (control), 10.8, 28.1 and 81.7μgSe/g dry mass. Embryos were reared under uncontaminated conditions until 50% of individuals within an aquarium had completed metamorphosis. The highest in ovo Se exposure group exhibited greater froglet body mass and snout to vent length while having a higher proportion of tadpoles at earlier stages of development. No differences were detected among treatment groups for mortality or metamorphic timing during the rearing period. This research suggests that in ovo Se exposure has minimal effect on the survival and development of late stage larval X. laevis in a controlled laboratory environment with adequate food availability.

Occurrence and the state of a tudor repeat protein in the development of Xenopus laevis

The Xenopus tudor repeat (Xtr) protein of the unfertilized and fertilized eggs at different intervals, and embryos of different stages was compared by immunoprecipitating with anti-Xtr monoclonal antibody, followed by Western blotting using anti-Xtr antibody. Besides the Xtr of 270 kDa, another band of Xtr below 150 kDa, was detected reproducibly after fertilization of the eggs. This result suggested that a part of Xtr was degraded after fertilization. The screening profile of the existence of Xtr and the distribution of degradation from unfertilized eggs to the nerula stage embryos showed that in the unfertilized eggs there was no occurrence of degradation. But it was visualized just after 20-minutes of fertilization and lasted up to the gastrula stage. Interestingly, the degradation could not be detected beyond this stage of development. Probably, the Xtr protein was degraded following the activation of the eggs because previous investigations suggested that Xtr acted after fertilization or activation of the eggs.Jahangirnagar University J. Biol. Sci. 3(2): 1-8, 2014 (December)

Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine

As a widely used herbicide, atrazine (AZ) has been extensively studied for its adverse effects on the reproductive system, especially feminization in male animals. However, the relationship of gene expression changes and associated toxicological endpoints remains unclear. In this study, developing Xenopus laevis tadpoles were exposed to concentration of AZ at 0.1, 1, 10 or 100 μg/L continuously. Compared with froglets in the control group, there were no significant differences in body length, body weight, liver weight and hepatosomatic index (HSI) of males in groups treated with AZ for 90 d. At 100 μg/L AZ treatment caused a significant reduction of gonad weight and gonadosomatic index (GSI) of males (p < 0.01). In addition, AZ at all dose levels caused testicular degeneration, especially in froglets from the groups with 0.1 and 100 μg/L which exhibited U-shaped dose-response trend. We further investigated the gene expression changes associated with the testicular degeneration induced by AZ. We found that the expression of 1165 genes was significantly altered with 616 upregulated and 549 downregulated compared to the expression profile of the control animals. KEGG analysis showed that genes which were significantly affected by AZ are mainly involved in arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesterone-mediated oocyte maturation pathway. Our results show that AZ may affect reproductive and immune systems by interference with the related gene expression changes during the male X. laevis development. The findings may help to clarify the feminization mechanisms of AZ in male X. laevis.

CUG-BP, Elav-like family member 1 (CELF1) is required for normal myofibrillogenesis, morphogenesis, and contractile function in the embryonic heart.

CUG-BP, Elav-like family member 1 (CELF1) is a multifunctional RNA binding protein found in a variety of adult and embryonic tissues. In the heart, CELF1 is found exclusively in the myocardium. However, the roles of CELF1 during cardiac development have not been completely elucidated. Myofibrillar organization is disrupted and proliferation is reduced following knockdown of CELF1 in cultured chicken primary embryonic cardiomyocytes. In vivo knockdown of Celf1 in developing Xenopus laevis embryos resulted in myofibrillar disorganization and a trend toward reduced proliferation in heart muscle, indicating conserved roles for CELF1 orthologs in embryonic cardiomyocytes. Loss of Celf1 also resulted in morphogenetic abnormalities in the developing heart and gut. Using optical coherence tomography, we showed that cardiac contraction was impaired following depletion of Celf1, while heart rhythm remained unperturbed. In contrast to cardiac muscle, loss of Celf1 did not disrupt myofibril organization in skeletal muscle cells, although it did lead to fragmentation of skeletal muscle bundles. CELF1 is required for normal myofibril organization, proliferation, morphogenesis, and contractile performance in the developing myocardium. Developmental Dynamics 245:854-873, 2016. © 2016 Wiley Periodicals, Inc.