The optic vesicle promotes cornea to lens transdifferentiation in larval Xenopus laevis

The outer cornea and pericorneal epidermis (lentogenic area) of larval Xenopus laevis are the only epidermal regions competent to regenerate a lens under the influence of the retinal inducer. However, the head epidermis of the lentogenic area can acquire the lens-regenerating competence following transplantation of an eye beneath it. In this paper we demonstrate that both the outer cornea and the head epidermis covering a transplanted eye are capable of responding not only to the retinal inducer of the larval eye but also to the inductive action of the embryonic optic vesicle by synthesizing crystallins. As the optic vesicle is a very weak lens inductor, which promotes crystallin synthesis only on the lens biased ectoderm of the embryo, these results indicate that the lens-forming competence in the outer cornea and epidermis of larval X. laevis corresponds to the persistence and acquisition of a condition similar to that of the embryonic biased ectoderm.     

Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that pose global ecological and human health problems. Although it is well established that PCBs are associated with a variety of adverse health effects in wildlife and in humans, it is often difficult to determine direct cause-and-effect relationships between exposure and specific health outcomes. In this study, gene expression signatures were used to relate exposure to PCBs with altered physiological responses and/or specific health effects. Real-time PCR was used to measure gene expression levels for 10 genes in Xenopus laevis tadpoles (18 days postfertilization, PF) after acute exposure (2 days) to the PCB mixture Aroclor 1254. Specific gene expression signatures correlated with exposure and were predictive of adverse health effects. Exposure to low levels of Aroclor 1254 (5-50 ppb) significantly increased expression of six genes, independent of any health effects; exposure to midlevel concentrations (300-400 ppb) significantly decreased expression levels of two genes, NGF and beta-actin, prior to the onset of observable health effects; exposure to higher doses (500-700 ppb) significantly decreased NGF and beta-actin expression concomitant with the appearance of gross morphological abnormalities, behavioral deficits, and a statistically significant decrease in survival. This study expands upon our previous work that demonstrated an age-dependent susceptibility to Aroclor 1254 in Xenopus laevis tadpoles and that defined specific gene expression signatures as useful bioindicators of exposure and as predictors of overt or impending health effects.     

Ultrastructural identification of the primitive muscle spindle in the Xenopus laevis larvae

Summary The initial formation of muscle spindles was studied with electron microscopy using the toe muscle of Xenopus laevis. At the larval stage 57 (Nieuwkoop and Faber 1967), muscle spindles were first identified primarily by the presence of sensory endings associated with a thin bundle of myotubes, e.g. intrafusal (IF) myotubes which were partly invested by a single cellular layer. The number of IF myotubes per spindle was 5 to 6; the adult complement. IF-and extrafusal (EF) myotubes were almost identical in their size and structure. A few thinner IF myotubes with scaree myofibrils were also present. The reticular zone had been undeveloped. Sensory endings were smaller in size and in number per spindle than those in the adult, forming irregular beaded chains with occasional tubular expansions. The endings and IF myotubes were rarely in direct contact, being frequently interposed by a satellite cell and its process. Incipient fusimotor endings were widely distributed from the juxta-equatorial to the polar region. Large cored vesicles resembling the neurosecretory vesicles occurred in sensory and motor endings as well as in intramuscular nerve fibers. The vesicles may be involved in the neuronal influence upon the spindle differentiation.The results were compared with the formative process of mammalian spindles.     

Aroclor 1254 alters morphology,survival, and gene expression in Xenopus laevis tadpoles

PCBs are persistent environmental contaminants that cause a variety of adverse health effects in wildlife and humans. This article describes the use of signature gene expression patterns that link increased PCB exposure with progressive, adverse biological effects. Developing Xenopus laevis tadpoles of two age classes were exposed to the PCB mixture Aroclor 1254 for 2 days. Real-time PCR was used to quantitate mRNA expression for 11 physiologically relevant, potential bioindicator genes. Younger tadpoles (5 days postfertilization) were resistant to Aroclor 1254 and showed few changes in gross morphology, swimming behavior, survival, or gene expression. Older tadpoles (11 days postfertilization) were more susceptible to Aroclor 1254. Exposure to 25 and 50 ppm Aroclor 1254 caused alterations in gross morphology and swimming behavior and statistically significant decreases in survival. These tadpoles showed statistically significant decreases in gene expression for 9 out of the 11 genes measured. Tadpoles exposed to 10 ppm showed incipient health changes but had gene expression profiles similar to the tadpoles treated with higher doses of Aroclor 1254. Tadpoles exposed to 1 ppm did not exhibit any observable adverse health effects, yet statistically significant decreases in gene expression occurred in these tadpoles (4 out of 11 genes). After prolonged exposure, tadpoles exposed to 1 and 10 ppm Aroclor 1254 exhibited health effects similar to those exposed to higher concentrations. Therefore, changes in expression of specific genes may serve not only as molecular bioindicators of Aroclor 1254 exposure but also as predictors of impending adverse health effects.     

Regeneration of Functional Pronephric Proximal Tubules After Partial Nephrectomy in Xenopus laevis

Background : While the renal system is critical for maintaining homeostatic equilibrium within the body, it is also susceptible to various kinds of damage. Tubule dysfunction in particular contributes to acute renal injury and chronic kidney disease in millions of patients worldwide. Because current treatments are highly invasive and often unavailable, gaining a better understanding of the regenerative capacity of renal structures is vital. Although the effects of various types of acute damage have been previously studied, the ability of the excretory system to repair itself after dramatic tissue loss due to mechanical damage is less well characterized. Results : A novel unilateral nephrectomy technique was developed to excise pronephric proximal tubules from Xenopus laevis tadpoles to study tubule repair after injury. Immunohistochemical detection of protein expression and renal uptake assays demonstrated that X. laevis larvae have the capacity to regenerate functional proximal tubules following resection. Conclusions : We have validated the renal identity of the restored tubules and demonstrated their ability to functional normally providing the first evidence of regeneration of renal tissue in an amphibian system. Importantly, this tubule restoration occurs by means of a process involving an early apoptotic event and the biphasic expression of the matrix metalloproteinase, Xmmp‐9. Developmental Dynamics 242:219–229, 2013. © 2012 Wiley Periodicals, Inc.     

Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis

In Xenopus laevis, two immunoglobulin light chain isotypes, designated L1 or ? and L2 or σ, have been identified. The genomic organization of the L1 locus has been described previously: a constant (C) gene segment is preceded by a joining (J) gene segment; in addition, there are many cross-hybridizing variable (V) gene segments. To evaluate the extent of sequence diversity of L1 V regions, we screened three cDNA libraries, constructed from mitogen-stimulated Xenopus splenocytes, with probes for the C or the J gene segment. Eighteen cDNA clones that contain complete or truncated V regions were chosen for sequence analysis. The C regions of all clones are identical or nearly identical to the genomic C gene segment; the V regions are greater than 80 % identical in nucleotide sequence and are presumably derived from a single family of V gene segments. Although framework regions are nearly identical, complementarity-determining regions are quite diverse. The expressed J segments fall into distinct groups, suggesting the presence of more than one germ-line J segment. Therefore, a genomic library was screened with a J region probe. A clone overlapping with the previously identified J- C clone, and containing four additional J gene segments, was isolated. All five J gene segments are very similar and three are identical in nucleotide sequence. Each of the three distinct germ-line J sequences is represented in the set of cDNA clones, suggesting that combinatorial diversification occurs; imprecision of V-J joining also appears to contribute to variability. Overall, these results suggest that the immunoglobulin repertoire in this species is not significantly restricted by a limitation in the diversity of light chain V regions.     

Restorative Regeneration of Digital Tips in the African Clawed Frog (Xenopus laevis Daudin)

Localized, specialized structures are carried on the tips of digits of many amniote and certain non‐amniote tetrapods. The pes of some members of Pipidae represents a rare example among tetrapods of differential expression of digital tip form (clawed vs. non‐clawed digits). As a step towards understanding how such localized forms are generated and maintained, we conducted a series of amputation experiments and observed, through the process of regeneration, the potential for reconstitution, at the tissue and organ level, of the different digit tip morphologies. Results of this study reveal that immediately following metamorphosis specialized digit tip structures are regenerated with a high degree of structural and spatial accuracy by a process that essentially replicates normal development in recently metamorphosed Xenopus laevis froglets. Furthermore, this regenerative capacity is maintained in juveniles 4 months beyond metamorphosis, and also in adults of 1 year or more in age, indicating that metamorphosis‐specific conditions do not exclusively facilitate regeneration of digit tips. In addition, regenerative capacity is maintained through repeated bouts of amputation and regeneration, indicating deep‐seated digit identity and retention of the distinct digit tip developmental programs within the digits. Together, these data suggest that the developmental programing responsible for the formation of the discrete digital tip morphologies is located regionally within each digit, and that it is retained through time. Our results suggest that Xenopus can serve as a model organism for exploring the molecular underpinnings of digit tip formation because regeneration leads to morphologically identical structures to those of the original digit tips. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Peroxisome biogenesis occurs in late dorsal-anterior structures in the development of Xenopus laevis.

Metabolism and development are two important processes not often examined in the same context. The focus of the present study is the expression of specific peroxisomal genes, the subsequent biogenesis of peroxisomes, and their potential role in the metabolism associated with the development of Xenopus laevis embryos. The temporal and expression patterns of six peroxisomal genes (PEX5, ACO, PEX19, PMP70, PEX16, and catalase) were elucidated using RT-PCR. Functionally related peroxisomal genes exhibited similar expression patterns with their RNA levels elevated relatively late during embryogenesis. Using immunohistochemistry PMP70 and catalase protein was localized largely to dorsal-anterior structures. Peroxisomal function was assayed with peroxisomal targeted-GFP, which when microinjected, revealed peroxisomes in dorsal-anterior structures at stage 45. A requirement for peroxisomal function appears to be present only late in development as organogenesis is finishing, yolk stores are depleted, and ingestion commences.     

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.     

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.     

Proper notch activity is necessary for the establishment of proximal cells and differentiation of intermediate,distal, and connecting tubule in Xenopus pronephros development

Background: Notch signaling in pronephros development has been shown to regulate establishment of glomus and proximal tubule, but how Notch signal works on competency of pronephric anlagen during the generation of pronephric components remains to be understood. Results: We investigated how components of pronephros (glomus, proximal tubule, intermediate tubule, distal tubule, and connecting tubule) were generated in Xenopus embryos by timed overactivation and suppression of Notch signaling. Notch activation resulted in expansion of the glomus and disruption of the proximal tubule formation. Inhibition of Notch signaling reduced expression of wt1 and XSMP‐30. In addition, when Notch signaling was overactivated at stage 20 on, intermediate, distal, and connecting tubule markers, gremlin and clcnkb, were decreased while Notch down‐regulation increased gremlin and clcnkb. Similar changes were observed with segmental markers, cldn19, cldn14, and rhcg on activation or inhibition of Notch. Although Notch did not affect the expression of pan‐pronephric progenitor marker, pax2, its activation inhibited lumen formation in the pronephros. Conclusions: Notch signal is essential for glomus and proximal tubule development and inhibition of Notch is critical for the differentiation of the intermediate, distal, and connecting tubule. Developmental Dynamics 245:472–482, 2016. © 2015 Wiley Periodicals, Inc.     

Expression profiles of the duplicated matrix metalloproteinase-9 genes suggest their different roles in apoptosis of larval intestinal epithelial cells during Xenopus laevis metamorphosis.

Matrix metalloproteinases (MMPs) play a pivotal role in development and/or pathogenesis through degrading extracellular matrix (ECM) components. We have previously shown that Xenopus MMP-9 gene is duplicated. To assess possible roles of MMP-9 and MMP-9TH in X. laevis intestinal remodeling, we here analyzed their expression profiles by in situ hybridization and show that their expression is transiently up-regulated during thyroid hormone-dependent metamorphosis. Of interest, MMP-9TH mRNA is strictly localized in the connective tissue and most highly expressed just beneath the larval epithelium that begins to undergo apoptosis. On the other hand, cells expressing MMP-9 mRNA become first detectable in the connective tissue and then, after the start of epithelial apoptosis, also in the larval epithelium. These results strongly suggest that MMP-9TH is responsible in the larval epithelial apoptosis through degrading ECM components in the basal lamina, whereas MMP-9 is involved in the removal of dying epithelial cells during amphibian intestinal remodeling.     

The thymus and tail regenerative capacity in Xenopus laevis tadpoles

A morphofunctional analysis of the thymus from differently aged Xenopus laevis tadpoles during regeneration of the tail is reported. In stage 50 larvae, competent to regenerate, the appendage cut provoked thymic structural modifications that affected the medullary microenvironment cells and changes in TNF-α immunoreactivity. Mucocyte-like cells, multicellular epithelial cysts, myoid cells and cells immunoreactive to TNF-α increased in number. Increased numbers of lymphocytes were also found in regenerating areas and, at the end of regeneration, thymic structural and immunocytochemical patterns were restored to control levels. The observed cellular responses and the induction of molecules critical for thymus constitutive processes suggest a stimulation of thymic function after tail amputation. In older larvae, whose capacity to form a new complete and correctly patterned tail was reduced, thymic morphological changes were more severe and may persist throughout the regeneration process with a significant reduction in organ size. In these larvae the histological patterns and the marked thymic decrease may be related to the events occurring during regeneration, i.e. the higher inflammatory response and the reduced tail regenerative potential.     

Wnt6 expression in epidermis and epithelial tissues during Xenopus organogenesis.

Here, we report the localization within embryonic tissues of xWnt6 protein; together with the temporal and spatial expression of Xenopus laevis Wnt6 mRNA. Wnt6 expression in Xenopus embryos is low until later stages of neurulation, when it is predominantly found in the surface ectoderm. Wnt6 expression increases during early organogenesis in the epidermis overlaying several developing organs, including the eye, heart, and pronephros. At later stages of development, Wnt6 mRNA and protein generally localize in epithelial tissues and specifically within the epithelial tissues of these developing organs. Wnt6 localization correlates closely with sites of both epithelial to mesenchymal transformations and mesenchymal to epithelial transformations. Xenopus Wnt6 sequence and its expression pattern are highly conserved with other vertebrates. Xenopus embryos, therefore, provide an excellent model system for investigating the function of vertebrate Wnt6 in organ development and regulation of tissue architecture.     

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.     

Development of the venous pole of the heart in the frog Xenopus laevis: A morphological study with special focus on the development of the venoatrial connections

The heart of lung‐breathing vertebrates normally shows an asymmetric arrangement of its venoatrial connections along the left‐right (L‐R) body axis. The systemic venous tributaries empty into the right atrium while the pulmonary venous tributaries empty into the left atrium. The ways by which this asymmetry evolves from the originally symmetrically arranged embryonic venous heart pole are poorly defined. Here we document the development of the venous heart pole in Xenopus laevis (stages 40–46). We show that, prior to the appearance of the mouth of the common pulmonary vein (MCPV), the systemic venous tributaries empty into a bilaterally symmetric chamber (sinus venosus) that is demarcated from the developing atriums by a circular ridge of tissue (sinu‐atrial ridge). A solitary MCPV appears during stage 41. From the time point of its first appearance onwards, the MCPV lies cranial to the sinu‐atrial ridge and to the left of the developing interatrial septum and body midline. L‐R lineage analysis shows that the interatrial septum and MCPV both derive from the left body half. The CPV, therefore, opens from the beginning into the future left atrium. The definitive venoatrial connections are established by the formation of a septal complex that divides the lumen of the venous heart pole into systemic and pulmonary venous flow pathways. This complex arises from the anlage of the interatrial septum and the left half of the sinu‐atrial ridge. Developmental Dynamics 240:1518–1527, 2011. © 2011 Wiley‐Liss, Inc.