In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification‐specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac‐Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac‐Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac‐Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.     

Temporal and spatial patterning of axial myotome fibers in Xenopus laevis

Somites give rise to the vertebral column and segmented musculature of adult vertebrates. The cell movements that position cells within somites along the anteroposterior and dorsoventral axes are not well understood. Using a fate mapping approach, we show that at the onset of Xenopus laevis gastrulation, mesoderm cells undergo distinct cell movements to form myotome fibers positioned in discrete locations within somites and along the anteroposterior axis. We show that the distribution of presomitic cells along the anteroposterior axis is influenced by convergent and extension movements of the notochord. Heterochronic and heterotopic transplantations between presomitic gastrula and early tail bud stages show that these cells are interchangeable and can form myotome fibers in locations determined by the host embryo. However, additional transplantation experiments revealed differences in the competency of presomitic cells to form myotome fibers, suggesting that maturation within the tail bud presomitic mesoderm is required for myotome fiber differentiation. Developmental Dynamics 239:1162–1177, 2010.© 2010 Wiley‐Liss, Inc.     

Appl1 is essential for the survival of Xenopus pancreas,duodenum, and stomach progenitor cells

An understanding of the molecular mechanisms governing the survival of organ progenitor cells in vivo is crucial for in vitro tissue regeneration. Here, we have found that Xenopus appl1 and akt2 share a similar embryonic expression pattern, showing characteristic expression in the central nervous system as well as in the pancreas and part of the stomach/duodenum (SD) at tadpole stages of development. Specific knockdown of appl1 in endoderm or inhibition of akt activity did not affect the formation of endodermal organ primordia at tail bud stages of development, but led to a gut‐coiling defect, strong apoptosis in endodermal organs, and pancreas and SD hypoplasia or even aplasia at tadpole stages of development. Furthermore, appl1 is required for akt phosphorylation and akt2 in turn can rescue appl1 knockdown phenotypes. Together, our data suggest that appl1‐akt signaling is specifically required for the survival of pancreas and SD progenitor cells in Xenopus laevis embryos. Developmental Dynamics 239:2198–2207, 2010. © 2010 Wiley‐Liss, Inc.     

Temporal and spatial patterning of axial myotome fibers in Xenopus laevis

An image of an X. laevis tadpole showing rhodamine‐labeled transplanted presomitic mesoderm cells (red) in which a subset of cells have differentiated into myotome fibers (shown in orange). The differentiated myotome fibers are stained with the antibody 12/101 (green) and the notochord is stained with Tor 70 (blue). The image was digitally duplicated twice and fused at the anterior end. From Krneta‐Stankic et al., Developmental Dynamics 239:1162–1177, 2010.     

Regulation of early Xenopus development by the PIAS genes

Originally identified as cytokine inhibitors, protein inhibitors of activated STAT (PIAS) are shown to regulate activities of a plethora of proteins and influence diverse processes such as immune response, cancer formation, and cell cycle progression. However, the roles of PIAS during vertebrate embryogenesis are less understood. In this study, we report isolation and initial characterization of all four PIAS genes from Xenopus laevis. The Xenopus PIAS genes are expressed throughout early development and have overlapping and distinct expression patterns, with, for example, high levels of PIAS2 in the notochord and strong expression of PIAS4 in the neural and neural crest derivatives. Overexpression of PIAS disrupts mesoderm induction and impairs body axis formation. PIAS proteins have differential ability to regulate signals from the growth factors activin, bone morphogenetic protein 4 (BMP4), and Wnt8. Our data suggest that Xenopus PIAS play important roles in mesodermal induction and patterning during early frog development. Developmental Dynamics 240:2120–2126, 2011. © 2011 Wiley‐Liss, Inc.     

Characterization of three synuclein genes in Xenopus laevis

The synuclein family consists of three small intracellular proteins mainly expressed in neural tissues, and has been associated with human neurodegenerative diseases. We have examined the spatial and temporal expression patterns of three synuclein genes during embryogenesis of Xenopus laevis. The Xenopus synucleins were firstly expressed in the developing nervous system at the tail bud stages. At tadpole stages, Xenopus snca was expressed in the brain, branchial arch and somite, and sncbb signals were detected in entire brain and spinal cord. However, sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion. RT‐PCR indicated that expression of synucleins was up‐regulated at the end of neurulation, and then maintained at later examined stages. Our study provides the spatiotemporal expression patterns of the synuclein family genes in Xenopus embryos, and forms a basis for further functional analysis of synucleins. Developmental Dynamics 240:2028–2033, 2011. © 2011 Wiley‐Liss, Inc.     

Identification of genes induced in regenerating Xenopus tadpole tails by using the differential display method.

To identify candidate gene(s) involved in the tail regeneration of Xenopus laevis tadpoles, we used the differential display method to isolate four genes (clones 1, 2, 13a, and 13b) whose expression is induced in regenerating tadpole tails. Among them, clones 13a and 13b were found to encode the Xenopus homologues of the alpha1 chain of type XVIII collagen and neuronal pentraxin I, respectively. Expression of clone 2 and neuronal pentraxin I genes increased dramatically in the blastema 3 days after amputation, whereas that for the clone 1 and type XVIII collagen genes was induced gradually after amputation. In situ hybridization revealed that the neuronal pentraxin I gene is expressed specifically in the regenerating tail epidermis but not in the normal tail epidermis or the most distal margin of the tail blastema, suggesting that it has a tissue-inductive role in tail regeneration. Expression of the four genes was induced in the limb and in the tail blastema, suggesting that they are involved in the regeneration of both organs. Finally, expression of clone 2 and neuronal pentraxin I genes was scarce during embryonic stages in comparison to the tail blastema, suggesting that their main functions are in organ regeneration. Our results demonstrate unique features of spatial and temporal gene expression patterns during Xenopus tadpole tail regeneration.     

KazrinA is required for axial elongation and epidermal integrity in Xenopus tropicalis.

Kazrin is a recently described desmosomal protein that binds the cornified envelope precursor periplakin. In this study, we have examined kazrin isoform A expression during the development of Xenopus tropicalis and investigated the consequences of its depletion. Whole mount in situ hybridisation revealed that kazrinA mRNA is expressed throughout the embryo at least until tadpole stages. Xenopus tropicalis embryos that had been injected with antisense morpholino oligonucleotides directed against kazrinA failed to elongate properly and showed defects in development of the head, eye, notochord, and somites. We also observed that the epidermis became disorganised and frequently separated from the underlying mesoderm, causing the formation of epidermal blisters. Together, our results suggest that loss of kazrinA causes defects in cell adhesion that affect axial elongation, cell differentiation, and epidermal morphogenesis.     

Sebox regulates mesoderm formation in early amphibian embryos

Background: Mix/Bix genes are important regulators of mesendoderm formation during vertebrate embryogenesis. Sebox, an additional member of this gene family, has been implicated in endoderm formation during early embryogenesis in zebrafish. However, it remains unclear whether Sebox plays a unique role in early Xenopus embryos. Results: In this study, we provide evidence that Sebox is uniquely required for the formation of mesoderm during early Xenopus embryogenesis. Sebox is dynamically expressed in the involuted mesoderm during gastrulation. It is activated by Nodal/Activin signaling and modulated by zygotic Wnt/β‐catenin signaling. Overexpression of Sebox perturbs movements during convergent extension and inhibits the expression of mesodermal, but not endodermal, genes induced by Nodal/Activin signaling. Depletion of Sebox using a specific morpholino increases the expression of noncanonical wnt5a, wnt5b, and wnt11b. Depletion of Sebox also up‐regulates the expression of pcdh8.2, a paraxial mesoderm‐specific protocadherin, in a Wnt11B‐dependent manner. Sebox morphants display reduced development of the head and notochord. Conclusions: Our findings illustrate that Sebox, a unique member of the Mix/Bix gene family, functions downstream of Nodal/Activin signaling and is required for the proper expression of noncanonical Wnt ligands and the normal development of mesoderm in Xenopus. Developmental Dynamics 244:1415–1426, 2015. © 2015 Wiley Periodicals, Inc.     

Cloning and characterization of voltage‐gated calcium channel alpha1 subunits in Xenopus laevis during development

Voltage‐gated calcium channels play a critical role in regulating the Ca²⁺ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore‐forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that Ca_V1.2, Ca_V2.1, Ca_V2.2, and Ca_V3.2 are expressed during neurula stages throughout the neural tube. These, along with Ca_V1.3 and Ca_V2.3, beginning at early tail bud stages, and Ca_V3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development. Developmental Dynamics 238:2891–2902, 2009. © 2009 Wiley‐Liss, Inc.     

Developmental expression of the fermitin/kindlin gene family in Xenopus laevis embryos

Fermitin genes are highly conserved and encode cytocortex proteins that mediate integrin signalling. Fermitin 1 (Kindlin1) is implicated in Kindler syndrome, a human skin blistering disorder. We report the isolation of the three Fermitin orthologs from Xenopus laevis embryos and describe their developmental expression patterns. Fermitin 1 is expressed in the skin, otic and olfactory placodes, pharyngeal arches, pronephric duct, and heart. Fermitin 2 is restricted to the somites and neural crest. Fermitin 3 is expressed in the notochord, central nervous system, cement gland, ventral blood islands, vitelline veins, and myeloid cells. Our findings are consistent with the view that Fermitin 1 is generally expressed in the skin, Fermitin 2 in muscle, and Fermitin 3 in hematopoietic lineages. Moreover, we describe novel sites of Fermitin gene expression that extend our knowledge of this family. Our data provide a basis for further functional analysis of the Fermitin family in Xenopus laevis. Developmental Dynamics 240:1958–1963, 2011. © 2011 Wiley‐Liss, Inc.     

Transgenic Analysis of Signaling Pathways Required for Xenopus Tadpole Spinal Cord and Muscle Regeneration

The Xenopus tadpole has the capacity fully to regenerate its tail after amputation. Previously, we have established that this regeneration process requires the operation of several signaling pathways including the bone morphogenic protein, Wnt, and Fgf pathways. Here, we have addressed the signaling requirements for spinal cord and muscle regeneration in a tissue‐specific manner. Two methods were used namely grafts of transgenic spinal cord to a wild type host, and the use of the Tet‐on conditional transgenic system to express inhibitors in the individual tissues. For the grafting experiments, the tail was amputated through the graft, which contained a temperature inducible inhibitor of the Wnt‐β‐catenin pathway. For the Tet‐on experiments, treatment with doxycycline was used to induce cell autonomous inhibitors of the Wnt‐β‐catenin or the Fgf pathway in either spinal cord or muscle. The results show that both spinal cord and muscle regeneration depend on both the Wnt‐β‐catenin and the Fgf pathways. This experimental design also enables us to observe the effect of inhibition of regeneration of one tissue on the regeneration of the others. Regardless of the method of inhibition, we find that reduction of spinal cord regeneration reduces regeneration of other parts of the tail, including the myotomal muscles. In contrast, reduction of muscle regeneration has no effect on the regeneration of the spinal cord. In common with other regeneration systems, this indicates that soluble factors from the spinal cord are needed to promote the regeneration of the other tissues in the tail. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Anterior‐posterior regionalized gene expression in the Ciona notochord

Background: In the simple ascidian chordate Ciona, the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non‐compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape, and behavior vary consistently along the anterior‐posterior (AP) axis. Results: Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non‐uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin‐like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF‐β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL, and TGF‐β expression are evident even before the notochord cells have intercalated into a single‐file column. Conclusions: We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. Developmental Dynamics 243:612–620, 2014. © 2013 Wiley Periodicals, Inc.     

Advancing genetic and genomic technologies deepen the pool for discovery in Xenopus tropicalis

Xenopus laevis and Xenopus tropicalis have long been used to drive discovery in developmental, cell, and molecular biology. These dual frog species boast experimental strengths for embryology including large egg sizes that develop externally, well-defined fate maps, and cell-intrinsic sources of nutrients that allow explanted tissues to grow in culture. Development of the Xenopus cell extract system has been used to study cell cycle and DNA replication. Xenopus tadpole tail and limb regeneration have provided fundamental insights into the underlying mechanisms of this processes, and the loss of regenerative competency in adults adds a complexity to the system that can be more directly compared to humans. Moreover, Xenopus genetics and especially disease-causing mutations are highly conserved with humans, making them a tractable system to model human disease. In the last several years, genome editing, expanding genomic resources, and intersectional approaches leveraging the distinct characteristics of each species have generated new frontiers in cell biology. While Xenopus have enduringly represented a leading embryological model, new technologies are generating exciting diversity in the range of discoveries being made in areas from genomics and proteomics to regenerative biology, neurobiology, cell scaling, and human disease modeling.     

Ouro proteins are not essential to tail regression during Xenopus tropicalis metamorphosis

Tail regression is one of the most prominent transformations observed during anuran metamorphosis. A tadpole tail that is twice as long as the tadpole trunk nearly disappears within 3 days in Xenopus tropicalis. Several years ago, it was proposed that this phenomenon is driven by an immunological rejection of larval‐skin‐specific antigens, Ouro proteins. We generated ouro‐knockout tadpoles using the TALEN method to reexamine this immunological rejection model. Both the ouro1‐ and ouro2‐knockout tadpoles expressed a very low level of mRNA transcribed from a targeted ouro gene, an undetectable level of Ouro protein encoded by a target gene and a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in tail skin. Furthermore, congenital athymic frogs were produced by Foxn1 gene modification. Flow cytometry analysis showed that mutant frogs lacked splenic CD8⁺ T cells, which play a major role in cytotoxic reaction. Furthermore, T‐cell‐dependent skin allograft rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles showed any significant delay in the process of tail shortening during the climax of metamorphosis, which shows that Ouro proteins are not essential to tail regression at least in Xenopus tropicalis and argues against the immunological rejection model.     

The Role of Sdf‐1α signaling in Xenopus laevis somite morphogenesis

Background: Stromal derived factor‐1α (sdf‐1α), a chemoattractant chemokine, plays a major role in tumor growth, angiogenesis, metastasis, and in embryogenesis. The sdf‐1α signaling pathway has also been shown to be important for somite rotation in zebrafish (Hollway et al., 2007). Given the known similarities and differences between zebrafish and Xenopus laevis somitogenesis, we sought to determine whether the role of sdf‐1α is conserved in Xenopus laevis. Results: Using a morpholino approach, we demonstrate that knockdown of sdf‐1α or its receptor, cxcr4, leads to a significant disruption in somite rotation and myotome alignment. We further show that depletion of sdf‐1α or cxcr4 leads to the near absence of β‐dystroglycan and laminin expression at the intersomitic boundaries. Finally, knockdown of sdf‐1α decreases the level of activated RhoA, a small GTPase known to regulate cell shape and movement. Conclusion: Our results show that sdf‐1α signaling regulates somite cell migration, rotation, and myotome alignment by directly or indirectly regulating dystroglycan expression and RhoA activation. These findings support the conservation of sdf‐1α signaling in vertebrate somite morphogenesis; however, the precise mechanism by which this signaling pathway influences somite morphogenesis is different between the fish and the frog. Developmental Dynamics 243:509–526, 2014. © 2013 Wiley Periodicals, Inc.