Xenopus Development Research Articles

On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development

A biochemical explanation of development from the fertilized egg to the adult requires an understanding of the proteins and RNAs expressed over time during embryogenesis. We present a comprehensive characterization of protein and mRNA dynamics across early development in Xenopus. Surprisingly, we find that most protein levels change little and duplicated genes are expressed similarly. While the correlation between protein and mRNA levels is poor, a mass action kinetics model parameterized using protein synthesis and degradation rates regresses protein dynamics to RNA dynamics, corrected for initial protein concentration. This study provides detailed data for absolute levels of ∼10,000 proteins and ∼28,000 transcripts via a convenient web portal, a rich resource for developmental biologists. It underscores the lasting impact of maternal dowry, finds surprisingly few cases where degradation alone drives a change in protein level, and highlights the importance of transcription in shaping the dynamics of the embryonic proteome.

A Database of microRNA Expression Patterns in Xenopus laevis.

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase.

Expression analysis of ciliary rootlet coiled coil protein mRNA during Xenopus development

Expression analysis of ciliary rootlet coiled coil protein mRNA during Xenopus development

Proteomics of Xenopus development.

Modern mass spectrometry-based methods provide an exciting opportunity to characterize protein expression in the developing embryo. We have employed an isotopic labeling technology to quantify the expression dynamics of nearly 6000 proteins across six stages of development in Xenopus laevis from the single stage zygote through the mid-blastula transition and the onset of organogenesis. Approximately 40% of the proteins show significant changes in expression across the development stages. The expression changes for these proteins naturally falls into six clusters corresponding to major events that mark early Xenopus development. A subset of experiments in this study have quantified protein expression differences between single embryos at the same stage of development, showing that, within experimental error, embryos at the same developmental stage have identical protein expression levels.

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Abstract 2995: Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer

Abstract Background: Despite scientific advances over the past decade, prostate cancer still remains the fifth leading cause of death from cancer in men worldwide. Docetaxel has an established role in the treatment of metastatic castrate-resistant prostate cancer (CRPC). However, malignant cells frequently acquire Docetaxel resistance. Therefore, further research is required to understand the molecular mechanisms underlying Docetaxel-resistance, which could be helpful in formulating alternative and superior therapeutic strategies. Methods: Two Docetaxel-resistant prostate cancer cell lines (IGR-CaP1 and PC3) were obtained by continuous exposure to Docetaxel. By comparing genes and miRs expression profiles established in these two Docetaxel-resistant models, we observed an enrichment of genes involved in the epithelial-to-mesenchymal transition (EMT) in the chemoresistant models and identified new genes potentially implicated in the resistance mechanism. Results: We identified the SHISA3 gene as a highly down-regulated gene in resistant cells. This gene was originally identified as an inhibitor of Wnt and FGF signaling during development in Xenopus. We showed that SHISA3 was lost during the acquisition of resistance to Docetaxel possibly via an EMT mechanism. Knockdown of SHISA3 in the parental IGR-CaP1 and PC3 cells triggered loss of the tight junction protein Occludin, engaged the Cadherin switch and increased the migratory properties of cells in vitro. Loss of SHISA3, along with loss of E-cadherin expression and expression of the mesenchymal marker N-cadherin were also observed in Docetaxel-resistant tumors obtained from IGR-CaP1-R xenografted mice. Phylogenetic comparison of SHISA3 gene sequences suggested that it may correspond to transmembrane adapters capable of regulating the activity of membrane receptors such as growth factor receptors. We are currently studying the binding partners of SHISA3 in our models by a proteomic approach to identify the implicated signaling pathway in prostate cancer. Conclusion: Our results show that loss of SHISA3 is an early event linked to the EMT process associated with chemoresistance and suggest that SHISA3 could be a useful biomarker to identify chemoresistant cells. Moreover, identification of its mechanism of action may lead to the identification of new therapeutic targets to overcome Docetaxel resistance. Citation Format: Nicolas J-p Martin, Sophie Cotteret, Catherine Gaudin, Marine Garrido, Safae Aarab-Terrisse, Nader al Nakouzi, Lucas Gentilini, Daniel Compagno, Vasily Ogryzko, Guillaume Meurice, Karim Fizazi, Anne Chauchereau. Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2995. doi:10.1158/1538-7445.AM2015-2995

Huntingtin is required for ciliogenesis and neurogenesis during early Xenopus development

Huntington's Disease (HD) is a neurodegenerative disorder that results from the abnormal expansion of poly-glutamine (polyQ) repeats in the Huntingtin (HTT) gene. Although HTT has been linked to a variety of cellular events, it is still not clear what the physiological functions of the protein are. Because of its critical role during mouse embryonic mouse development, we investigated the functions of Htt during early Xenopus embryogenesis. We find that reduction of Htt levels affects cilia polarity and function and causes whole body paralysis. Moreover, Htt loss of function leads to abnormal development of trigeminal and motor neurons. Interestingly, these phenotypes are partially rescued by either wild-type or expanded HTT. These results show that the Htt activity is required for normal embryonic development, and highlight the usefulness of the Xenopus system for investigating proteins involved in human diseases.

Transcriptional regulator PRDM12 is essential for human pain perception.

Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP). Here we identified 10 different homozygous mutations in PRDM12 (encoding PRDI-BF1 and RIZ homology domain-containing protein 12) in subjects with CIP from 11 families. Prdm proteins are a family of epigenetic regulators that control neural specification and neurogenesis. We determined that Prdm12 is expressed in nociceptors and their progenitors and participates in the development of sensory neurons in Xenopus embryos. Moreover, CIP-associated mutants abrogate the histone-modifying potential associated with wild-type Prdm12. Prdm12 emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics.

Developmental role of plk4 in Xenopus laevis and Danio rerio: implications for Seckel Syndrome.

The polo-like kinases are a family of conserved serine/threonine kinases that play multiple roles in regulation of the cell cycle. Unlike its four other family members, the role of Plk4 in embryonic development has not been well characterized. In mice, Plk4(-)(/)(-) embryos arrest at E7.5, just prior to the initiation of somitogenesis. This has led to the hypothesis that Plk4 expression may be essential to somitogenesis. Recently characterized human mutations lead to Seckel Syndrome. Riboprobe in situ hybridization revealed that plk4 is ubiquitously expressed during early stages of development of Xenopus and Danio; in later stages, expression in frogs restricts to somites as well as eye, otic vesicle, and branchial arch, and brain. Expression patterns in fish remain ubiquitous. Both somite and eye development require planar cell polarity, and disruption of plk4 function in frog by means of morpholino-mediated translational knockdown yields orientational disorganization of both these structures. These results provide the first steps in defining a new role for plk4 in organogenesis and implies a role in planar cell polarity, segmentation, and in recently described PLK4 mutations in human.

Subchronic and chronic developmental effects of copper oxide (CuO) nanoparticles on Xenopus laevis

Metal oxide nanoparticles, such as copper oxide (CuO), are mass produced for use in a variety of products like coatings and ceramics. Acute exposure to CuO nanoparticles has caused toxicity to many aquatic organisms, yet there is no information on the effect of prolonged CuO nanomaterial exposures. This study examined effects of chronic exposure to CuO nanoparticles on Xenopus laevis growth and development. Experiments included a 14d subchronic exposure and a 47d chronic exposure throughout metamorphosis. The subchronic exposure caused mortality in all tested CuO concentrations, and significant growth effects occurred after exposure to 2.5mgL−1 CuO. Chronic exposure to 0.3mgL−1 CuO elicited significant mortality and affected the rate of metamorphosis. Exposure to lower concentrations of CuO stimulated metamorphosis and growth, indicating that low dose exposure can have hormetic effects.

The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.

The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.

Maternal Wnt/STOP signaling promotes cell division during early Xenopus embryogenesis

During Xenopus development, Wnt signaling is thought to function first after midblastula transition to regulate axial patterning via β-catenin-mediated transcription. Here, we report that Wnt/glycogen synthase kinase 3 (GSK3) signaling functions posttranscriptionally already in mature oocytes via Wnt/stabilization of proteins (STOP) signaling. Wnt signaling is induced in oocytes after their entry into meiotic metaphase II and declines again upon exit into interphase. Wnt signaling inhibits Gsk3 and thereby protects proteins from polyubiquitination and degradation in mature oocytes. In a protein array screen, we identify a cluster of mitotic effector proteins that are polyubiquitinated in a Gsk3-dependent manner in Xenopus. Consequently inhibition of maternal Wnt/STOP signaling, but not β-catenin signaling, leads to early cleavage arrest after fertilization. The results support a novel role for Wnt signaling in cell cycle progression independent of β-catenin.

ATP4 and ciliation in the neuroectoderm and endoderm of Xenopus embryos and tadpoles.

During gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/β-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012) 516–527.) and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015)) of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/β-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/β-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development.

RNA and the synapse.

RNA and the synapse.

Developmental expression analysis of Na, K-ATPase α subunits in Xenopus.

Na, K-ATPase is an integral membrane protein complex responsible for maintaining the ionic gradients of Na(+) and K(+) across the plasma membrane and has a variety of cellular functions including neuronal activity. Studies in several organisms have shown that this protein complex regulates multiple aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Here, we report the cloning and expression of Na, K-ATPase α2 (atp1a2) and α3 (atp1a3) subunits during Xenopus development and compare the expression patterns of each subunit. Using in situ hybridization in whole embryos and on sections, we show that all three α subunits are co-expressed in the pronephric kidney, with varying expression in neurogenic derivatives. The atp1a2 has a unique expression in the ependymal cell layer of the developing brain that is not shared with other α subunits. The Na, K-ATPase α1 (atp1a1), and atp1a3 share many expression domains in placode derivatives, including the otic vesicle, lens, ganglion of the anterodorsal lateral line nerve, and ganglia of the facial and anteroventral lateral line nerve and olfactory cells. All the subunits share a common expression domain, the myocardium.

The ribosome biogenesis factor Nol11 is required for optimal rDNA transcription and craniofacial development in Xenopus.

The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC) of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival.

Spatiotemporal expression of Prdm genes during Xenopus development.

Epigenetic regulation is known to be important in embryonic development, cell differentiation and regulation of cancer cells. Molecular mechanisms of epigenetic modification have DNA methylation and histone tail modification such as acetylation, phosphorylation and ubiquitination. Until now, many kinds of enzymes that modify histone tail with various functional groups have been reported and regulate the epigenetic state of genes. Among them, Prdm genes were identified as histone methyltransferase. Prdm genes are characterized by an N-terminal PR/SET domain and C-terminal some zinc finger domains and therefore they are considered to have both DNA-binding ability and methylation activity. Among vertebrate, fifteen members are estimated to belong to Prdm genes family. Even though Prdm genes are thought to play important roles for cell fate determination and cell differentiation, there is an incomplete understanding of their expression and functions in early development. Here, we report that Prdm genes exhibit dynamic expression pattern in Xenopus embryogenesis. By whole mount in situ hybridization analysis, we show that Prdm genes are expressed in spatially localized manners in embryo and all of Prdm genes are expressed in neural cells in developing central nervous systems. Our study suggests that Prdm genes may be new candidates to function in neural cell differentiation.

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

SUMMARYNeural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Distinct expression patterns and functions of the pygopus genes in amphioxus and zebrafish early embryogenesis

The Wnt/β-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machine to β-catenin to regulate the transcription of target genes. Previous studies revealed that the Pygo genes were broadly involved in the embryonic development of Drosophila, Xenopus and mouse, however, their functions in the embryonic development of amphioxus and zebrafish remain to be investigated. Here, we cloned and characterized one Pygo gene from amphioxus Branchiostoma belcheri (BbPygo) and two from zebrafish Danio rerio (DrPygo1 and DrPygo2). The spatial–temporal expression profiles showed that all these genes were maternally expressed. BbPygo expression was distributed ubiquitously in the amphioxus embryos before the gastrula stage and became restricted in the pharyngeal region in late stages. Similar to BbPygo, DrPygo2 was also expressed throughout whole embryos before the early gastrula stage, but limited in the pharyngeal region and central nervous system (CNS) in subsequent developing stages. Further knockdown of the DrPygo2 resulted in developmental defects of the body axis, CNS and cartilage segments in zebrafish. In contrast, the expression signal of the DrPygo1 gene was not detectable using the in situ hybridization method, which is consistent with its extremely low expression level detected by RT-qPCR. The expression patterns and function investigations revealed in this study suggest that the three Pygo genes probably function differentially though all of them show a conserved maternal expression pattern during the early embryonic stages of amphioxus and zebrafish.

A novel function for Egr4 in posterior hindbrain development.

Segmentation of the vertebrate hindbrain is an evolutionarily conserved process. Here, we identify the transcription factor early growth response 4 (egr4) as a novel regulator of posterior hindbrain development in Xenopus. egr4 is specifically and transiently expressed in rhombomeres 5 and 6 (r5/r6), and Egr4 knockdown causes a loss of mafb/kreisler and krox20/egr2 expression in r5/r6 and r5, respectively. This phenotype can be fully rescued by injection of frog or mouse Egr4 mRNA. Moreover Egr4-depleted embryos exhibit a specific loss of the neural crest stream adjacent to r5, and have inner ear defects. While the homeodomain protein vHnf1/Hnf1b directly activates Mafb and Krox20 expression in the mouse hindbrain to specify r5, we show that in Xenopus this process is indirect through the activation of Egr4. We provide evidence that rearrangements in the regulatory sequences around egr4 and mafb genes may account for this difference.