Transdifferentiation of ocular tissues in larval Xenopus laevis

Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well.     

Regenerative responses in cultured hindlimb stumps of larval Xenopus laevis.

The regenerative capacity of larval Xenopus laevis hindlimbs amputated through the tarsalia at different stages of development and explanted in vitro was tested. In the first experimental series hindlimb stumps from stage 53, 54, 55, and 57 larvae (according to Nieuwkoop and Faber, '56) were cultured in Leibovitz's L-15 medium supplemented with 10% FCS, and 0.04 U of insulin and 10(-8) mg of L-thyroxine per ml of medium. Results showed that the distal part of the limb stumps from stages 53, 54, and 55 formed a regeneration blastema composed of proliferating mesenchymal cells beneath a typical apical cap. No blastema was formed in the proximal part of the stump. In limb stumps from stage 57, a regeneration blastema did not form either in the proximal or in the distal part of the stump. In a second experimental series, hindlimb stumps from stage 55 larvae, denervated 5 days prior to amputation in order to eliminate any residual neurotrophic factor, were cultured in a simplified L-15 medium containing 2% FCS and lacking insulin and thyroxine. Results showed that also in these experimental conditions the stumps from stage 55 formed a conical regeneration blastema at the distal tip. The blastema cells duplicated their own DNA and divided. At the proximal extremity no regeneration blastema was formed. In the same culture medium, the stumps of larvae at stage 57 did not form a regeneration blastema.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xapelin and Xmsr are required for cardiovascular development in Xenopus laevis

The cardiovascular development is the elaborate process, and despite the extensive studies, the mechanisms underlying endothelial, hematopoietic, and cardiac developments, as well as the interrelation between these processes, are not fully understood. In this study, we demonstrated that Xenopus apelin and Xmsr play pivotal roles in cardiovascular development. Apelin is a recently identified ligand for an orphan G-protein-coupled receptor APJ and is involved in fluid homeostasis in mammals. Xenopus preproapelin (Xpreproapelin) was isolated and its mRNA localized to the region around the presumptive blood vessels, which are overlapping or adjacent to those expressing Xmsr, the Xenopus homologue of APJ. Overexpression of Xpreproapelin disorganized the expression of the endothelial precursor cell marker XlFli and the hematopoietic precursor cell marker SCL at the neurula, whereas embryos injected with morpholino antisense oligonucleotides for Xapelin and Xmsr displayed attenuated expression of Tie2, alpha-globin, XPOX2, and cTnI, markers of endothelium, erythrocytes, myeloid cells, and cardiomyocytes, respectively. XlFli morpholino had similar effects to Xapelin and Xmsr morpholinos on cardiac differentiation, suggesting an unexpected potential relationship between the endothelium and cardiac differentiation. Forced expression of constitutive active G alpha i rescued the phenotypes of Xmsr morpholino-injected embryos, indicating that the i/o type of G protein alpha subunit acts downstream of Xmsr.     

Nerve-independent DNA synthesis and mitosis in regenerating hindlimbs of larval Xenopus laevis

Summary Xenopus laevis larvae at stage 52–53 (according to Nieuwkoop and Faber 1956) were subjected to amputation of both limbs at the thigh level as well as to repeated denervations of the right limb. Results obtained in larvae sacrificed during wound healing (1 after amputation), blastema formation (3 days) and blastema growth (5 and 7 days) showed that denervated right limbs have undergone the same histological modifications observed in innervated left limbs and have formed a regeneration blastema consisting of mesenchymal cells with a pattern of DNA synthesis and mitosis very similar to that in presence of nerves. Also, the patterns of cellular density in regenerating right and left limbs were very similar. On the whole, the data here reported show a highly remarkable degree of nerve-independence for regeneration in hindlimbs of larval Xenopus laevis at stage 52–53 and lend some substance to the hypothesis that, in early limbs, there would exist trophic factors capable of replacing those released by nerves, promoting DNA synthesis and mitosis in blastemal cells. Offprint requests to: S. Filoni     

Translational control of activin in Xenopus laevis embryos

Activin is a potent mesoderm inducing factor present in embryos of Xenopus laevis. Recent evidence has implicated activin in the inhibition of neural development in addition to the well-established induction of mesoderm in ectodermal explants. These diverse effects are critically dependent on the concentration of activin yet little is known about the mechanisms regulating the level of activin in the embryo. We report that the 3′ untranslated region (3′ UTR) of activin βB mRNA inhibits the translation of activin in embryos. Microinjection of activin mRNA from which the 3′ UTR has been deleted is 8–10-fold more potent in inducing mesoderm than mRNA containing the 3′ UTR. Truncation of the 3′ UTR also leads to a marked enhancement of activin protein levels in embryos but has no effect when the truncated mRNA is translated in vitro. The 3′ UTR also confers translational inhibition on a heterologous mRNA. These data show that a maternal factor(s) present in X. laevis regulates the translation of injected activin βB mRNA. This factor(s) could be responsible for regulating the levels of endogenous activin βB protein during mesoderm induction and the specification of ectodermal derivatives such as neural and epidermal tissues. © 1995 Wiley-Liss, Inc.     

Maternal control of pattern formation in Xenopus laevis

We review the essential role of maternal factors in pattern formation for Xenopus laevis, focusing on VegT, Vg1, and Wnt11. Results from loss of function experiments demonstrate a clear requirement for these genes in germ layer specification, dorsal-ventral axis formation, and convergence extension. We also discuss these genes in the broader context of metazoan development, exploring whether and how their functions in the X. laevis model organism may or may not be conserved in other species. Wnt11 signaling in particular provides a classic example where understanding context in development is crucial to understanding function. Genomic sequencing, gene expression, and functional screening data that are becoming available in more species are providing invaluable aid to decoding and modeling signaling pathways. More work is needed to develop a comprehensive catalog of the Wnt signaling, T-box, and TGF-beta genes in metazoans both near and far in evolutionary distance. We finally discuss some specific experimental and modeling efforts that will be needed to understand the behavior of these signaling networks in vivo so that we can interpret these critical pathways in an evolutionary framework.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.     

Tissue interactions and lens-forming competence in the outer cornea of larval Xenopus laevis

After lentectomy through the pupillary hole, the outer cornea of larval Xenopus laevis can undergo transdifferentiation to regenerate a new lens. This process is elicited by inductive factor(s) produced by the neural retina and accumulated into the vitreous chamber. During embryogenesis, the outer cornea develops from the outer layer of the presumptive lens ectoderm (PLE) under the influence of the eye cup and the lens. In this study, we investigated whether the capacity of the outer cornea to regenerate a lens is the result of early inductive signals causing lens-forming bias and lens specification of the PLE, or late inductive signals causing cornea formation or both signals. Fragments of larval epidermis or cornea developed from ectoderm that had undergone only one kind of inductive signals, or both kinds of signals, or none of them, were implanted into the vitreous chamber of host larvae. The regeneration potential and the lens-forming transformations of the implants were tested using an antisense probe for pax6 as an earlier marker of lens formation and a monoclonal antibody anti-lens as a definitive indicator of lens cell differentiation. Results demonstrated that the capacity of the larval outer cornea to regenerate a lens is the result of both early and late inductive signals and that either early inductive signals alone or late inductive signals alone can elicit this capacity.     

Development of adrenergic and cholinergic cardiac control in larvae of the African clawed frog Xenopus laevis

Cardiac responses (heart rate, stroke volume, and cardiac output) to cholinergic and adrenergic receptor stimulation were investigated in developing larvae of Xenopus laevis from Nieuwkoop and Faber (NF) stage 33/34 (newly hatched) to NF stage 53 (22 d after hatching). Effects on heart rate (fH), stroke volume (SV), and cardiac output (CO) were analyzed using in situ preparations and video-microscopic techniques to record the continually beating heart. The results show that administration of acetylcholine to the heart decreases heart rate as early as NF stage 40. A significant reduction in SV and CO following acetylcholine administration to the heart was found at NF stages 45-53. Epinephrine had no significant effect on fH, SV, or CO at any of the stages investigated. However, an adrenergic tonus on the heart is present already at NF stage 40 (11%). This tonus increases up to a maximum (44%) at NF stages 45-47, when the maximal heart rate is found during development of X. laevis. We conclude that acetylcholine has a negative chronotropic and possibly also inotropic effect on the heart very early in development of X. laevis. We also hypothesize that the high adrenergic tonus found at NF stages 45-47 is responsible, at least in part, for the peak in heart rate seen at these stages.     

Intrinsic control of regenerative loss in Xenopus laevis limbs

The regenerative capability of Xenopus laevis hindlimbs was studied at different developmental stages. Three types of surgical deletion of the autopod were performed: simple amputations, 3-digit wedge-shaped deletions and 1-digit wedge-shaped deletions. The frequency of regenerative response and the digital patterns of the regenerates were analyzed. Regenerative capacity declines with developmental stage for all three types of deletions. For simple amputations this decline occurs in an orderly manner with the failure of anterior digits to regenerate at progressively later stages. A comparison between 1-digit and 3-digit deletions shows that for all stages of development, 3-digit deletions regenerate better than one-digit deletions. These data indicate that the amount of tissue removed is directly related to whether a regenerative response is observed. At any given stage, larger deletions are more likely to regenerate than smaller ones. These results are discussed with regard to the mechanisms by which growth during regeneration is controlled.     

Assessment of TRH as a potential MSH release stimulating factor in Xenopus laevis

This study considers the possible involvement of the tripeptide TRH (thyrotropin releasing hormone) in the physiological regulation of melanophore stimulating hormone (MSH) secretion from the pars intermedia of the toad, Xenopus laevis. TRH was shown to stimulate release of MSH from superfused neurointermediate lobes obtained from white-background adapted animals, but had no effect on secretion from lobes of black-background adapted animals. Immunohistochemical analysis revealed a rich TRH-containing neuronal network terminating in the neural lobe of the Xenopus pituitary. Plasma levels of TRH, determined with a specific radioimmunoassay, proved to be extremely high and no significant difference in this level could be found between white- and black-adapted animals. Plasma TRH probably originates from the skin, and our results show that its concentration is within the effective concentration range established for this peptide in stimulating MSH release from the pars intermedia. Therefore, while both our superfusion and immunohistochemical results argue favourably for a function of TRH in the regulation of MSH secretion, we conclude that, in any regulatory role, it would likely have to function within the pars intermedia at concentrations exceeding the high plasma values. While TRH could be involved in short-term activation of the secretory process in white-background adapted animals or in animals undergoing the initial stages of black background adaptation, our results indicate that this peptide may have no function in the maintenance of secretion from the pars intermedia of animals fully adapted to black background.     

Evidence for a mitochondrial chromosome in Xenopus laevis oocytes

When Xenopus laevis mitochondria are gently lysed at physiologic ionic strength, mitochondrial DNA (mitDNA) is extracted associated with proteins. Sedimentation and buoyant density studies indicate that proteins are bound to mitDNA at a ratio of about 1/1. This DNA-protein complex visualized by electron microscopy after fixation with glutaraldehyde appears as a relaxed circular molecule consisting of an average of 48 globular particles interconnected by a thin filament.