Developing Xenopus Laevis Embryos Research Articles

Calcium dynamics at the neural cell primary cilium regulate Hedgehog signaling–dependent neurogenesis in the embryonic neural tube

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.

Developmental exposure to the pesticide malathion enhances expression of Prdm12, a regulator of nociceptor development, in Xenopus laevis.

The transcription factor Prdm12 exerts important influences on the development of nociceptors, peripheral touch and pain-sensing neurons, and has been implicated in human pain sensation disorders. We examined the consequences of exposing developing Xenopus laevis embryos to the commonly used pesticide malathion on Prdm12 expression. Using qPCR and western blot analysis we observed that malathion treatment for the first six days of tadpole development significantly increased both prdm12 mRNA levels and Prdm12 protein levels compared to controls. Consequently, early exposure to this pesticide has potential to alter nociceptor development.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Lethal and Teratogenic Impacts of Imazapyr, Diquat Dibromide, and Glufosinate Ammonium Herbicide Formulations Using Frog Embryo Teratogenesis Assay-Xenopus (FETAX).

Globally, amphibians are experiencing widespread abnormalities and population declines. One potential contributor to these challenges is the use of pesticides, particularly aquatic herbicides applied to aquatic habitats inhabited by amphibians. Critical issues of concern are the potential toxicity and teratogenicity of these herbicides towards amphibians. Using the FETAX protocol, three globally used formulations, including diquat dibromide (Midstream), glufosinate ammonium (Basta), and imazapyr (Arsenal), were assessed for embryotoxicity, teratogenicity, and growth inhibition. Developing Xenopus laevis embryos were exposed for 96h atconcentrations of 0.5-3.0mg/L, 1.6-3.0mg/L, and 20-45mg/L for Midstream, Basta, and Arsenal respectively. The 96-h LC50 estimates were 0.83mg/L acid equivalent (a.e.), 36mg/L a.e., and 2.2mg/L a.e., whereas the EC50 estimates were 0.24mg/L a.e., 28.13mg/L a.e., and 2.01mg/L a.e. for the Midstream, Arsenal, and Basta formulations, respectively. These two estimates produced Teratogenic Index of 3.5, 1.3, and 1.1 for Midstream, Arsenal, and Basta, respectively, indicating a high risk of malformation induction by Midstream and moderate risk for Arsenal. Regarding growth inhibition, lowest observable effect concentrations of 0.5mg/L, 25mg/L, and 2.0mg/L were computed for Midstream, Arsenal, and Basta, respectively, producing the minimum concentration inhibiting growth (MCIG) ratios of 0.62, 0.69, and 0.89 for the three formulations. These MICG values are higher than the standard 0.30 growth inhibitors benchmark, suggesting that the formulations are not growth inhibitors at the evaluated concentrations. This study provides evidence of the embryotoxic and teratogenic status of Midstream and the embryotoxicity of Basta. There is a need to further characterise the physiological and ecological impacts of these formulations to ensure responsible use and the safety of amphibians and other wildlife.

The effect of vaping on the development and craniofacial morphology of Xenopus laevis embryos.

Vaping is a type of nicotine usage in which an artificial cigarette is used as the delivery system. Originally marketed as the healthier alternative to smoking cigarettes, vaping has led to an increase in the amount of pulmonary issues by users. Our objective for this research was to test whether the consumption of nicotine and flavor additives through vaping is as harmful to developing embryos as that obtained through smoking traditional cigarettes. For this research, we exposed developing Xenopus laevis embryos to nicotine from vaping solution with 3 differing propylene glycol: vegetable glycerin ratios (30:70, 50:50, 70:30) as well as smoke from a well-known cigarette brand, all of which contain 12 mg of nicotine. We exposed the embryos to the same amount of nicotine and byproducts that would be consumed by the average pregnant American woman who either vapes or smokes traditional cigarettes. Embryos were exposed from Stage 10 (yolk plug) to Stage 43 (freeswimming tadpole with most yolk consumed). Tadpoles were sacrificed and mounted, and Image J was used to determine if there were any differences in craniofacial morphology between treatments. Preliminary results are encouraging and indicate possible developmental effects of different VG: PG ratios. This research was funded by a NASA Space Grant awarded to Camden Cutlip.

LOC496300 is expressed in the endoderm of developing Xenopus laevis embryos.

LOC496300 is expressed in the endoderm of developing Xenopus laevis embryos.

Temporal control of growth factor‐mediated signaling pathways during cell differentiation and Xenopus embryonic development

Growth factor‐mediated signaling pathways regulate a wide‐spectrum of cellular functions such as survival, proliferation, differentiation, and apoptosis. Different growth factors typically trigger the activation of the same set of downstream signaling pathways which includes the mitogen activated protein kinase (MAPK), phosphoinositide 3‐kinase (PI3K)‐AKT, and phospholipase C (PLC) pathways Evidence suggests that precise spatiotemporal regulation of these signaling pathways is involved in generating diverse cellular outcomes. Although conventional genetic and pharmacological approaches have helped us understand different aspects of signaling pathways, a quantitative delineation of their signaling kinetics is pending due to a lack of tools that allow for precise temporal control of these pathways. Non‐neuronal optogenetics, an emerging technique that utilizes light to control intracellular signaling pathways, is a remarkable tool to help achieve this goal. Here, we introduce a bicistronic optogenetic system which we recently developed in our laboratory to achieve temporal control of the MAPK pathway. This tool uses the photoactivatable protein cryptochrome 2 (CRY2) and the N‐terminal domain of cryptochrome‐interacting basic‐helix‐loop‐helix (CIBN) from Arabidopsis thaliana. This novel system allows reversible, light‐mediated activation of the MAPK signaling pathway in intact mammalian cells as well as in developing Xenopus laevis embryos. In PC12 neuronal cells, light‐controlled, intermittent MAPK activity reveals a memory effect in light‐induced neurite outgrowth. In Xenopus embryos, developmental stage‐specific MAPK activation further reveals that this pathway can reprogram cell fate after a crucial time window when germ layers are specified. Our strategy can be generalized to control other kinase pathways with a similar activation mechanism. Results from our research will help resolve intracellular mechanisms of growth factor‐mediated signal transduction during cell differentiation and embryonic development.Support or Funding InformationKai Zhang, Department of Biochemistry, University of Illinois Urbana‐ChampaignThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Control Neurotrophin Signaling using Light during PC12 Cell Differentiation and Xenopus Embryonic Development

Growth factor-mediated signaling pathways regulate neuronal survival, proliferation, differentiation, and apoptosis. The mitogen-activated protein kinase (MAPK) signaling pathway is a primary signaling cascade downstream of the binding of neurotrophins to their receptors. Evidence suggests that signaling output of the MAPK pathway varies with its temporal kinetics. A quantitative delineation of signaling kinetics is limited due to a lack of tools that allows precise temporal control of the MAPK pathway. The emerging non-neuronal optogenetics, which utilizes light to control intracellular signaling pathways, provides a new modality for spatiotemporal signaling control. We have developed an optogenetic system that allows reversible activation of the MAPK signaling pathway in intact cells and in developing Xenopus laevis embryos. In PC12 neuronal cell lines, light-controlled, intermittent MAPK activity reveals a memory effect in light-induced neurite outgrowth. In Xenopus embryos, developmental stage-specific MAPK activation implies that this pathway can reprogram cell fate after germ layer specification, a crucial time window during which destination of cell fate is set. Our strategy can be generalized to control other kinase pathways with a similar activation mechanism. Results from our research will help resolve intracellular mechanisms of neurotrophin-regulated signal transduction during cell differentiation and embryonic development.

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.

Assessing differences in toxicity and teratogenicity of three phthalates, Diethyl phthalate, Di-n-propyl phthalate, and Di-n-butyl phthalate, using Xenopus laevis embryos

ABSTRACTPhthalates, compounds used to add flexibility to plastics, are ubiquitous in the environment. In particular, the diethyl (DEP), di-n-propyl (DnPP), and di-n-butyl (DBP) phthalates were found to exert detrimental effects in both mammalian and non-mammalian studies, with toxic effects varying according to alkyl chain length. Embryos of Xenopus laevis, the African clawed frog, have been used to assess toxicity and teratogenicity of several compounds and serves as a model for assessing adverse and teratogenic effects of ortho-phthalate esters. The purpose of this study was to develop a model for comparison of developmentally toxic effects of ortho-phthalate esters using Xenopus embryos. In this study developing Xenopus laevis embryos were exposed to increasing concentrations of DEP, DnPP, and DBP using the 96-h Frog Embryo Teratogenesis Assay–Xenopus (FETAX), with 96-h lethal concentrations, effective concentrations to induce malformations, teratogenic indices, and concentrations to inhibit growth determined. DEP, DnPP, and DBP showed enhanced toxicity with increasing ester length. Developing Xenopus laevis exposed to DEP, DnPP, and DBP showed similar malformations that also occurred at lower concentrations with increasing alkyl chain length. Teratogenic risk did not change markedly with alkyl chain length, with data showing only DBP to be teratogenic.

Effects of 17β-estradiol, nonylphenol, and bisphenol-A on developing Xenopus laevis embryos

Many chemicals released into the environment have the capacity to disrupt the normal development of aquatic animals. We investigated the influence of nonylphenol (NP), bisphenol-A (BPA), and 17β-estradiol (E2) on developing Xenopus laevis embryos, as a model animal in the aquatic environment. Embryos were exposed to eight different concentrations of NP, BPA or E2 between 3 and 96 h post-fertilization (p.f.). Short body length, microcephaly, flexure, edema, and abnormal gut coiling were induced by 20 μM NP, BPA or 10 μM E2 by 96 h p.f. To clarify sensitive stages to these compounds, embryos were exposed to chemicals for 45 or 48 h starting at different developmental stages and experiments were terminated 96 h p.f. BPA and NP induced abnormalities in developing X. laevis, though the sensitive stages of embryos to these chemicals are different, BPA affecting earlier stages and NP affecting at later stages. To analyze the functional mechanisms of BPA and NP in induction of morphological changes, we adapted a DNA array technology and identified 6 X. laevis genes, XIRG, α skeletal tropomyosin, cyclin G1, HGF, troponin C2, and ribosomal protein L9. These findings may provide important clues to elucidate common mechanisms underlying teratogenic effects of these chemicals.

How do linker histones mediate differential gene expression?

In developing Xenopus laevis embryos the multiple-copy oocyte-type 5S RNA genes are progressively shut down. Results presented in three recent articles 1-3 together demonstrate that replacement of the cleavage stage linker histone B4 by somatic H1 leads to chromatosomes positioned directly over these genes and adjacent sequences so as to occlude the binding site for the critical transcription factor TFIIIA. In contrast, on the somatic-type 5S genes the somatic H1 positions chromatosomes about 65 bp further upstream, thereby leaving the TFIIIA binding site exposed and the genes active. The somatic linker histone thus functions as a specific gene repressor.

Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus.

Expression of transgenes within a single generation by direct DNA injection into vertebrate embryos has been plagued by inefficient and nonuniform gene expression. We report a novel strategy for efficient and stable expression of transgenes driven by both ubiquitous and tissue-specific promoters by direct DNA injection into developing Xenopus laevis embryos. This strategy involves flanking expression cassettes of interest with inverted terminal repeat sequences (ITRs) from adeno-associated virus. Our results suggest that the ITR strategy may be generally applicable to other systems, such as zebra fish and embryonic stem cells, and may enable tissue-specific expression of transgenes in problematic contexts.

NeuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family.

We have identified two new genes, neuroD2 and neuroD3, on the basis of their similarity to the neurogenic basic-helix-loop-helix (bHLH) gene neuroD. The predicted amino acid sequence of neuroD2 shows a high degree of homology to neuroD and MATH-2/NEX-1 in the bHLH region, whereas neuroD3 is a more distantly related family member. neuroD3 is expressed transiently during embryonic development, with the highest levels of expression between days 10 and 12. neuroD2 is initially expressed at embryonic day 11, with persistent expression in the adult nervous system. In situ and Northern (RNA) analyses demonstrate that different regions of the adult nervous system have different relative amounts of neuroD and neuroD2 RNA. Similar to neuroD, expression of neuroD2 in developing Xenopus laevis embryos results in ectopic neurogenesis, indicating that neuroD2 mediates neuronal differentiation. Transfection of vectors expressing neuroD and neuroD2 into P19 cells shows that both can activate expression through simple E-box-driven reporter constructs and can activate a reporter driven by the neuroD2 promoter region, but the GAP-43 promoter is preferentially activated by neuroD2. The noncongruent expression pattern and target gene specificity of these highly related neurogenic bHLH proteins make them candidates for conferring specific aspects of the neuronal phenotype.

Transgenic engineering of neuromuscular junctions in Xenopus laevis embryos transiently overexpressing key cholinergic proteins.

To examine the role of key cholinergic proteins in the formation of neuromuscular junctions (NMJs), we expressed DNAs encoding the mouse muscle nicotinic acetylcholine receptor (nAChR) or human brain and muscle acetylcholinesterase (hAChE) in developing Xenopus laevis embryos. Acetylthiocholine hydrolysis and alpha-bungarotoxin binding in homogenates of transgenic embryos revealed transient overexpression of the respective proteins for at least 4 days postfertilization. Moreover, hAChE injection induced an approximately 2-fold increase in endogenous Xenopus nAChR. Electron microscopy coupled with cytochemical staining for AChE activity revealed that AChE-stained areas, which reached 0.17 microns2 in NMJs of control embryos raised at 21 degrees C, increased up to 0.53 and 0.60 microns2 in nAChR and hAChE transgenics, respectively. These increases coincided with the appearance of a class of large NMJs with average postsynaptic lengths up to 1.8-fold greater than controls. As much as 57% and 34% of the NMJs in animals transgenic for nAChR and hAChE, respectively, displayed AChE activity in nerve terminals in addition to muscle labeling, as compared with 10% nerve-labeled NMJs in control animals. Moreover, area, but not length values, were > 2-fold larger in hAChE-expressing NMJs labeled in their nerve terminals than in those labeled in muscle alone, reflecting a hAChE-induced increase in synaptic cleft width. These findings indicate that modulation of cholinergic neurotransmission in NMJs modifies the features of nerve-muscle connections.

Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant.

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.

Spatial and temporal expression of phosphorylated and non-phosphorylated forms of neurofilament proteins in the developing nervous system of Xenopus laevis

Immunocytochemical studies of developing Xenopus laevis embryos and tadpoles (stages 12 1 2 to 46 ) were performed using a panel of 11 monoclonal antibodies to phosphorylated and non-phosphorylated forms of the neurofilament proteins. These included nine antibodies to the middle molecular weight neurofilament protein (XNF-M, 175 kDa), and two additional antibodies to non-phosphorylated forms of the other two neurofilament proteins (XNF-L, 73 kDa; XNF-H, 205 kDa). The developmental expression of XNF-M, XNF-L and XNF-H, and the progressive phosphorylation of XNF-M in the rhombencephalon, spinal cord, and optic nerve were studied using these antibodies. In the spinal cord and rhombencephalon, non-phosphorylated forms of XNF-M were initially detected during neural tube stages (stages 22–26), one day before XNF-L and XNF-H at early tadpole stages (stage 35/36). In the eye, XNF-M was observed initially during tailbud stages (stage 29/30), but neither XNF-L nor XNF-H was seen even by stage 46 (swimming tadpole). The phosphorylation of XNF-M occurred over a protracted period of several days, both in the neural tube and visual system, and could be divided into four phases. (1) When initially expressed, XNF-M was hypophosphorylated. This was indicated by the early immunostaining of axons and cell bodies with antibodies to dephosphorylated epitopes on XNF-M and by the absence of staining with antibodies to phosphorylated epitopes. (2) After a short timelag (3–9 h) axons were stained by some, but not all antibodies to phosphorylated epitopes. (3) Approximately one day later, all antibodies to phosphorylated epitopes stained the relevant axons. However, XNF-M was not yet fully phosphorylated, as indicated by the continued staining of these axons with antibodies to dephosphorylated epitopes of XNF-M. (4) Two to 3 days after the initial expression of XNF-M, dephosphorylated epitopes disappeared from the axons, establishing the adult pattern. During development, the most heavily phosphorylated neurofilament proteins present at a given stage were found first in distal regions of the axons and progressed gradually toward the neuronal perikarya as development proceeded. This gradient of phosphorylation, established early within the axon, suggests that neurofilaments in the axons mature from their distal ends toward the cell body, a process which may be regulated by local factors within the axons themselves. The similarity of the basic features of NF-M phosphorylation in mammalian, avian, and amphibian axons underscores the importance of this phenomenon for the development of a mature axon.

Effects of nabam on developing Xenopus laevis embryos: minimum concentration, biological stability, and degradative products.

This study was carried out to determine 1) the minimum concentration at which the fungicide ethylenebisdithiocarbamate disodium salt (nabam) would induce malformations in developingXenopus laevis embryos, 2) whether nabam in solution retained toxicological and teratogenic effects over long periods of time, 3) to compare the toxic and teratogenic effects of nabam's degradative products ethylene thiourea and ethylene diamine, 4) to compare toxic and teratogenic effects of the related substances methyl isothiocyanate and sodium thiocyanate, and 5) to compare the effects of combinations of these substances.Xenopus laevis embryos at “yolk plug” stage (Nieukoop and Faber stages 10–12) were exposed to various concentrations of fresh and one- to seven-week old nabam solutions, ethylene thiourea, ethylene diamine, methyl isothiocyanate, sodium thiocyanate, and combinations of some of these substances. Fresh nabam solutions induced malformations of the notochord in a majority of embryos at concentrations as low as 40 Μg/L. Embryos treated with 40 Μg/L nabam stored 1–7 weeks developed normally. Embryos treated with 100 Μg/L nabam stored 1–7 weeks developed severe malformations of the notochord, delayed pigmentation of the eye, and increased mortality. In addition, embryos exposed to 100 Μg/L nabam stored 4–7 weeks also experienced delayed development of the gut and a general decrease in size. Embryos exposed to fresh 100 Μg/L nabam solutions developed only malformations of the notochord. Concentrations of methyl isothiocyanate of less than 1.0 Μg/L were sublethal. Methyl isothiocyanate (1.0–1,000 Μg/L) was extremely toxic with 100% mortality recorded by day 7 in all groups. Sodium thiocyanate, ethylene thiourea, and ethylene diamine (100–10,000 Μg/L) were neither toxic nor teratogenic. Combinations of ethylene thiourea and methyl isothiocyanate at or beyond 5,000 Μg/L, or 1.0 Μg/L respectively were acutely lethal. At concentrations of 100–1,000 Μ/L ethylene thiourea and 0.01–0.1 Μg/L methyl isothiocyanate, the tadpoles that hatched had swollen and kinked notochords, similar to animals exposed to fresh 40 Μg/L nabam. Thus, the aquatic environment when contaminated with very low levels of the fungicide nabam is hazardous to the development of amphibian embryos.

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis

The changes in the average cAMP and cGMP content of developing Xenopus laevis embryos have been studied. Both nucleotides exhibited complicated patterns of change. It is possible, however, to correlate the changes in the ratio between the nucleotides during the early phases of development with the current notion that a low ratio is a distinction of mitotically active cells.