Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up‐regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan‐Trk inhibitor K252a or the TrkA inhibitor GW‐441756, but not by the TrkB inhibitor ANA‐12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.     

Xenopus cDNA microarray identification of genes with endodermal organ expression.

The endoderm is classically defined as the innermost layer of three Metazoan germ layers. During organogenesis, the endoderm gives rise to the digestive and respiratory tracts as well as associated organs such as the liver, pancreas, and lung. At present, however, how the endoderm forms the variety of cell types of digestive and respiratory tracts as well as the budding organs is not well understood. In order to investigate the molecular basis and mechanism of organogenesis and to identify the endodermal organ-related marker genes, we carried out microarray analysis using Xenopus cDNA chips. To achieve this goal, we isolated the Xenopus gut endoderm from three different stages of Xenopus organogenesis, and separated each stage of gut endoderm into anterior and posterior regions. Competitive hybridization of cDNA between the anterior and posterior endoderm regions, to screen genes that specifically expressed in the major organs, revealed 915 candidates. We then selected 104 clones for in situ hybridization analysis. Here, we report the identification and expression patterns of the 104 Xenopus endodermal genes, which would serve as useful markers for studying endodermal organ development.     

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.     

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.     

Ontogeny of central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui

Embryonic development of the central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, was determined by using immunocytochemistry. The majority of anuran amphibians (frogs) possess a larval stage (tadpole) that undergoes metamorphosis, a dramatic post-embryonic event, whereby the tadpole transforms into the adult phenotype. Directly developing frogs have evolved a derived life-history mode where the tadpole stage has been deleted and embryos develop directly into the adult bauplan. Embryonic development in E. coqui is classified into 15 stages (TS 1–15; 1 = oviposition / 15 = hatching). Serotonergic immunoreactivity was initially detected at TS 6 in the raphe nuclei in the developing rhombencephalon. At TS 7, immunopositive perikarya were observed in the paraventricular organ in the hypothalamus and reticular nuclei in the hindbrain. Development of the serotonergic system was steady and gradual during mid-embryogenesis. However, starting at TS 13 there was a substantial increase in the number of serotonergic neurons in the paraventricular, raphe, and reticular nuclei, a large increase in the number of varicose fibers, and a differentiation of the reticular nuclei in the hindbrain. Consequentially, E. coqui displayed a well-developed central serotonergic system prior to hatching (TS 15). In comparison, the serotonergic system in metamorphic frogs typically starts to develop earlier but the surge of development that transpires in this system occurs post-embryonically, during metamorphosis, and not in the latter stages of embryogenesis, as it does in E. coqui. Overall, the serotonergic development in E. coqui is similar to the other vertebrates.     

Photoreceptor Development in Premetamorphic and Metamorphic Xenopus laevis

Transgenic Xenopus laevis are commonly used to study gene expression in photoreceptors, but only red rods and red cones are known to exist in the pre‐metamorphic stages commonly used in transgenic studies. Using RT‐PCR, this study shows that violet cones develop in early pre‐metamorphic stages (Stage 35) with the red rods and red cones. Green rod development began in Stage 53 with the onset of metamorphosis. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Localization of endogenous galactoside-binding lectin during morphogenesis ofXenopus laevis

Summary A monoclonal antibody has been produced againstXenopus laevis galactoside-binding neural-creststage lectin. This antibody inhibits lectin-mediated hemagglutination. Using this antibody in conjunction with immunohistochemical techniques, lectin deposition has been studied in embryos and tadpoles at different stages of morphogenesis, from initial neural crest migration, up to the formation of a swimming tadpole. Lectin levels change during development in different regions of the embryo and tadpole, decreasing in migratory cells, and increasing in sites where cells become more adhesive to one another. The results suggest that galactoside-binding lectins may be an important class of cellular adhesion molecules during these stages of development.     

Sox17 and chordin are required for formation of Kupffer's vesicle and left‐right asymmetry determination in zebrafish

Kupffer's vesicle (KV), a ciliated fluid‐filled sphere in the zebrafish embryo with a critical role in laterality determination, is derived from a group of superficial cells in the organizer region of the gastrula named the dorsal forerunner cells (DFC). We have examined the role of the expression of sox17 and chordin (chd) in the DFC in KV formation and laterality determination. Whereas sox17 was known to be expressed in DFC, its function in these cells was not studied before. Further, expression of chd in these cells has not been reported previously. Targeted knockdown of Sox17 and Chd in DFC led to aberrant Left‐Right (L‐R) asymmetry establishment, as visualized by the expression of southpaw and lefty, and heart and pancreas placement in the embryo. These defects correlated with the formation of small KVs with apparently diminished cilia, consistent with the known requirement for ciliary function in the laterality organ for the establishment of L‐R asymmetry. Developmental Dynamics 239:2980–2988, 2010. Published 2010 Wiley‐Liss, Inc.     

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.     

Prolonged in vivo imaging of Xenopus laevis

Background: While live imaging of embryonic development over long periods of time is a well established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages, continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. Results: Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS‐222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis, by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution. Conclusions: This automated imaging system provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days. Developmental Dynamics 243:1011–1019, 2014. © 2014 Wiley Periodicals, Inc.     

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

Background: 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. Results: 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. Conclusions: 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.     

Emx3 is required for the differentiation of dorsal telencephalic neurons

emx3 is first expressed in prospective telencephalic cells at the anterior border of the zebrafish neural plate. Knockdown of Emx3 function by morpholino reduces the expression of markers specific to dorsal telencephalon, and impairs axon tract formation. Rescue of both early and late markers requires low‐level expression of emx3 at the one‐ or two‐somite stage. Higher emx3 expression levels cause dorsal telencephalic markers to expand ventrally, which points to a possible role of emx3 in specifying dorsal telencephalon and a potential new function for Wnt/beta‐catenin pathway activation. In contrast to mice, where Emx2 plays a major role in dorsal telencephalic development, knockdown of zebrafish Emx2 apparently does not affect telencephalic development. Similarly, Emx1 knockdown has little effect. Previously, emx3 was thought to be fish‐specific. However, we found all three emx orthologs in Xenopus tropicalis and opossum (Monodelphis domestica) genomes, indicating that emx3 was present in an ancestral tetrapod genome. Developmental Dynamics 238:1984–1998, 2009. © 2009 Wiley‐Liss, Inc.