Comparing the effects of three neonicotinoids on embryogenesis of the South African clawed frog Xenopus laevis.

Neonicotinoids (NEOs) are widely used insecticides that are ubiquitous in agricultural use. Since NEOs are found in natural waters as well as in tap water and human urine in regions where NEOs are widely used, NEOs pose a potential hazard to non-target organisms such as animals and humans. Some of the commonly detected NEOs are imidacloprid (IMD), thiamethoxam (TMX), and its metabolite clothianidin (CLO). Although previously published scientific information, including an assessment of the environmental risks, particularly for bees, had resulted in a ban on the outdoor use of these three NEOs in the EU - their use is now only permitted in closed greenhouses - these NEOs continue to be used in agriculture in many other parts of the world. Therefore, a detailed study and comparison of the effects of NEOs on the embryonic development of non-target organisms is needed to further define the risk profiles. Embryos of the South African clawed frog Xenopus laevis, a well-established aquatic model, were exposed to different concentrations of IMD, TMX, or CLO (0.1-100 mg/L) to study and compare the possible effects of a single contaminant in natural water bodies on early embryogenesis. The results included a reduced body length, a smaller orbital space, impaired cranial cartilage and nerves, and an altered heart structure and function. At the molecular level, NEO exposure partially resulted in an altered expression of tissue-specific factors, which are involved in eye, cranial placode, and heart development. Our results suggest that the NEOs studied negatively affect the embryonic development of the non-target organism X. laevis. Since pesticides, especially NEOs, pollute the environment worldwide, it is suggested that they are strictly controlled and monitored in the areas where they are used. In addition, the question arises as to whether pesticide metabolites also pose a risk to the environment and need to be investigated further so that they can be taken into account when registering ingredients.

The impact of the insecticide acetamiprid on the embryogenesis of the aquatic model organism Xenopus laevis.

Acetamiprid (ACT) is used extensively in agriculture worldwide, although data on ACT concentrations in natural water bodies and its impact on aquatic organisms are limited. To study whether ACT influences the embryogenesis of the South African clawed frog Xenopus laevis, embryos were incubated in ACT solutions from 0.01 to 100 mg/L. The low concentrations were chosen on the basis of concentrations already found in nature. ACT treatment leads to shorter embryo lengths, intestine malformation and reduced eye areas. It also affects the cranial cartilage and cardiac development as well as the embryo's mobility. The expression of tissue-specific marker genes is affected as well. Thus, our study suggests that pesticides may lead to an increased mortality of non-target organisms and emphasizes the importance of regular testing for ACT concentrations in nature. Our study provides an overview of ACT effects and can therefore be used as a basis for an ACT risk assessment.

Glyphosate without Co-formulants affects embryonic development of the south african clawed frog Xenopus laevis.

BACKGROUND: Glyphosate (GLY) is the most widely used herbicide in the world. Due to its mode of action as an inhibitor of the 5-enolpyruvylshikimate-3-phosphate synthase, an important step in the shikimate pathway, specifically in plants, GLY is considered to be of low toxicity to non-target organisms. However, various studies have shown the negative effects of GLY on the mortality and development of different non-target organisms, including insects, rodents, fish and amphibians. To better understand the various effects of GLY in more detail, we studied the effects of GLY without co-formulants during the embryogenesis of the aquatic model organism Xenopus laevis. RESULTS: A treatment with GLY affected various morphological endpoints in X. laevis tadpoles (body length, head width and area, eye area). Additionally, GLY interfered with the mobility as well as the neural and cardiac development of the embryos at stage 44/45. We were able to detect detailed structural changes in the cranial nerves and the heart and gained insights into the negative effects of GLY on cardiomyocyte differentiation. CONCLUSION: The application of GLY without co-formulants resulted in negative effects on several endpoints in the early embryonic development of X. laevis at concentrations that are environmentally relevant and concentrations that reflect the worst-case scenarios. This indicates that GLY could have a strong negative impact on the survival and lives of amphibians in natural waters. As a result, future GLY approvals should consider its impact on the environment.

The neonicotinoid thiacloprid leads to multiple defects during early embryogenesis of the South African clawed frog (Xenopuslaevis).

There is increasing concern about the health effects of pesticides that pollute natural waters. In particular, the use of neonicotinoids, such as thiacloprid (THD), is causing unease. THD is considered non-toxic to non-target vertebrates. Studies classify THD as carcinogenic, toxic to reproduction, and therefore harmful to the environment. A detailed study of possible THD effects during the amphibian embryogenesis is needed because leaching can introduce THD into aquatic environments. We incubated stage 2 embryos of the South African clawed frog in various THD concentrations (0.1-100 mg/L) at 14 °C to study the potential effects of a one-time THD contamination of waters on the early embryogenesis. We showed that THD has, indeed, negative effects on the embryonic development of the X. laevis. A treatment with THD led to a reduced embryonic body length and mobility. Furthermore, a treatment with THD resulted in smaller cranial cartilages, eyes and brains, and the embryos had shorter cranial nerves and an impaired cardiogenesis. On a molecular basis, THD led to a reduced expression of the brain marker emx1 and the heart marker mhcα. Our results underly the importance of a strict and efficient monitoring of the regulatory levels and application areas of THD.

Functions of block of proliferation 1 during anterior development in Xenopus laevis.

Block of proliferation 1 (Bop1) is a nucleolar protein known to be necessary for the assembly of the 60S subunit of ribosomes. Here, we show a specific bop1 expression in the developing anterior tissue of the South African clawed frog Xenopus laevis. Morpholino oligonucleotide-mediated knockdown approaches demonstrated that Bop1 is required for proper development of the cranial cartilage, brain, and the eyes. Furthermore, we show that bop1 knockdown leads to impaired retinal lamination with disorganized cell layers. Expression of neural crest-, brain-, and eye-specific marker genes was disturbed. Apoptotic and proliferative processes, which are known to be affected during ribosomal biogenesis defects, are not hindered upon bop1 knockdown. Because early Xenopus embryos contain a large store of maternal ribosomes, we considered if Bop1 might have a role independent of de novo ribosomal biogenesis. At early embryonic stages, pax6 expression was strongly reduced in bop1 morphants and synergy experiments indicate a common signaling pathway of the two molecules, Bop1 and Pax6. Our studies imply a novel function of Bop1 independent of ribosomal biogenesis.

The Ribosomal Protein L5 Functions During Xenopus Anterior Development Through Apoptotic Pathways.

Ribosomal biogenesis is a fundamental process necessary for cell growth and division. Ribosomal protein L5 (Rpl5) is part of the large ribosomal subunit. Mutations in this protein have been associated with the congenital disease Diamond Blackfan anemia (DBA), a so called ribosomopathy. Despite of the ubiquitous need of ribosomes, clinical manifestations of DBA include tissue-specific symptoms, e.g., craniofacial malformations, eye abnormalities, skin pigmentation failure, cardiac defects or liver cirrhosis. Here, we made use of the vertebrate model organism Xenopus laevis and showed a specific expression of rpl5 in the developing anterior tissue correlating with tissues affected in ribosomopathies. Upon Rpl5 knockdown using an antisense-based morpholino oligonucleotide approach, we showed different phenotypes affecting anterior tissue, i.e., defective cranial cartilage, malformed eyes, and microcephaly. Hence, the observed phenotypes in Xenopus laevis resemble the clinical manifestations of DBA. Analyses of the underlying molecular basis revealed that the expression of several marker genes of neural crest, eye, and brain are decreased during induction and differentiation of the respective tissue. Furthermore, Rpl5 knockdown led to decreased cell proliferation and increased cell apoptosis during early embryogenesis. Investigating the molecular mechanisms underlying Rpl5 function revealed a more than additive effect between either loss of function of Rpl5 and loss of function of c-Myc or loss of function of Rpl5 and gain of function of Tp53, suggesting a common signaling pathway of these proteins. The co-injection of the apoptosis blocking molecule Bcl2 resulted in a partial rescue of the eye phenotype, supporting the hypothesis that apoptosis is one main reason for the phenotypes occurring upon Rpl5 knockdown. With this study, we are able to shed more light on the still poorly understood molecular background of ribosomopathies.

Retinol binding protein 1 affects Xenopus anterior neural development via all-trans retinoic acid signaling.

BACKGROUND: Retinol binding protein 1 (Rbp1) acts as an intracellular regulator of vitamin A metabolism and retinoid transport. In mice, Rbp1 deficiency decreases the capacity of hepatic stellate cells to take up all-trans retinol and sustain retinyl ester stores. Furthermore, Rbp1 is crucial for visual capacity. Although the function of Rbp1 has been studied in the mature eye, its role during early anterior neural development has not yet been investigated in detail. RESULTS: We showed that rbp1 is expressed in the eye, anterior neural crest cells (NCCs) and prosencephalon of the South African clawed frog Xenopus laevis. Rbp1 knockdown led to defects in eye formation, including microphthalmia and disorganized retinal lamination, and to disturbed induction and differentiation of the eye field, as shown by decreased rax and pax6 expression. Furthermore, it resulted in reduced rax expression in the prosencephalon and affected cranial cartilage. Rbp1 inhibition also interfered with neural crest induction and migration, as shown by twist and slug. Moreover, it led to a significant reduction of the all-trans retinoic acid target gene pitx2 in NCC-derived periocular mesenchyme. The Rbp1 knockdown phenotypes were rescued by pitx2 RNA co-injection. CONCLUSION: Rbp1 is crucial for the development of the anterior neural tissue.

Nosip functions during vertebrate eye and cranial cartilage development.

BACKGROUND: The nitric oxide synthase interacting protein (Nosip) has been associated with diverse human diseases including psychological disorders. In line, early neurogenesis of mouse and Xenopus is impaired upon Nosip deficiency. Nosip knockout mice show craniofacial defects and the down-regulation of Nosip in the mouse and Xenopus leads to microcephaly. Until now, the exact underlying molecular mechanisms of these malformations were still unknown. RESULTS: Here, we show that nosip is expressed in the developing ocular system as well as the anterior neural crest cells of Xenopus laevis. Furthermore, Nosip inhibition causes severe defects in eye formation in the mouse and Xenopus. Retinal lamination as well as dorso-ventral patterning of the retina were affected in Nosip-depleted Xenopus embryos. Marker gene analysis using rax, pax6 and otx2 reveals an interference with the eye field induction and differentiation. A closer look on Nosip-deficient Xenopus embryos furthermore reveals disrupted cranial cartilage structures and an inhibition of anterior neural crest cell induction and migration shown by twist, snai2, and egr2. Moreover, foxc1 as downstream factor of retinoic acid signalling is affected upon Nosip deficiency. CONCLUSIONS: Nosip is a crucial factor for the development of anterior neural tissue such the eyes and neural crest cells. Developmental Dynamics 247:1070-1082, 2018. © 2018 Wiley Periodicals, Inc.

Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development.

Developmental neurogenesis in mouse and Xenopus is impaired in the absence of Nosip.

BACKGROUND: Genetic deletion of Nosip in mice causes holoprosencephaly, however, the function of Nosip in neurogenesis is currently unknown. RESULTS: We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of Nosip in neurogenesis. We found, that size and cortical thickness of the developing brain of Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of Rbp1, a regulator of retinoic acid synthesis, when Nosip was absent. We identified the homologous nosip gene to be expressed in differentiated neurons in the developing brain of Xenopus embryos. Knockdown of Nosip in Xenopus resulted in a reduction of brain size that could be rescued by reintroducing human NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon Nosip depletion. In Xenopus Nosip and Rbp1 are similarly expressed and knockdown of Nosip resulted in down regulation of Rbp1. Knockdown of Rbp1 caused a similar microcephaly phenotype as the depletion of Nosip and synergy experiments indicated that both proteins act in the same signalling pathway. CONCLUSIONS: Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of Rbp1 during early neurogenesis.

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.

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.

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation.

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/ß-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/ß-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.

Comparative expression study of sipa family members during early Xenopus laevis development.

The signal-induced proliferation-associated (SIPA) protein family belongs to the RapGAP protein superfamily. Previous studies mainly focused on the expression and function of SIPA genes in vertebrate neuronal tissue. Only limited data about the embryonic expression pattern of the genes are currently available. Our study provides the first expression analysis of sipa1, sipa1l1, sipa1l2, and sipa1l3 during early development of the vertebrate organism Xenopus laevis. In silico, analysis revealed that all genes are highly conserved across species. Semi-quantitative RT-PCR experiments demonstrated that the RNA of all genes was maternally supplied. By whole mount in situ hybridization approaches, we showed that sipa1 is mainly expressed in various sensory organs, the respiratory and blood system, heart, neural tube, and eye. In contrast, sipa1l1 showed a broad expression during development in particular within the brain, somites, eye, and heart. Sipa1l2 was detected in the branchial arches, glomerulus, and the developing eye. In contrast, sipa1l3 revealed a tissue specific expression within the olfactory and otic vesicles, the cranial placodes and ganglia, neural tube, pronephros, retina, and lens. In summary, all sipa gene family members are expressed throughout the whole developing Xenopus organism and might play an important role during vertebrate early embryogenesis.