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As a member of the seven-transmembrane rhodopsin-like G protein-coupled receptor superfamily, the melanocortin-3 receptor (MC3R) is vital for the regulation of energy homeostasis and rhythms synchronizing in mammals, and its pharmacological effect could be directly influenced by the presence of melanocortin receptor accessory proteins (MRAPs), MRAP1 and MRAP2. The tetrapod amphibian Xenopus laevis (xl) retains higher duplicated genome than extant teleosts and serves as an ideal model system for embryonic development and physiological studies. However, the melanocortin system of the Xenopus laevis has not yet been thoroughly evaluated. In this work, we performed sequence alignment, phylogenetic tree, and synteny analysis of two xlMC3Rs. Co-immunoprecipitation and immunofluorescence assay further confirmed the co-localization and in vitro interaction of xlMC3Rs with xlMRAPs on the plasma membrane. Our results demonstrated that xlMRAP2.L/S could improve α-MSH-stimulated xlMC3Rs signaling and suppress their surface expression. Moreover, xlMC3R.L showed a similar profile on the ligands and surface expression in the presence of xlMRAP1.L. Overall, the distinct pharmacological modulation of xlMC3R.L and xlMC3R.S by dual MRAP2 proteins elucidated the functional consistency of melanocortin system during genomic duplication of tetrapod vertebrates.

Melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R), two neural G protein-coupled receptors are known to be functionally critical for energy balance in vertebrates. As allosteric regulators of melanocortin receptors, melanocortin accessory proteins (MRAPs) are also involved in energy homeostasis. The interaction of MRAPs and melanocortin signaling was previously shown in mammals and zebrafish, but nothing had been reported in amphibians. As the basal class of tetrapods, amphibians occupy a phylogenetic transition between teleosts and terrestrial animals. Here we examined the evolutionary conservation of MC3R, MC4R, and MRAPs between diploid Xenopus tropicalis (xt-) and other chordates and investigated the pharmacological regulatory properties of MRAPs on the neural MC3R and MC4R signaling. Our results showed that xtMRAP and xtMRAP2 both exerted robust potentiation effect on agonist (α-MSH and adrenocorticotropin [ACTH]) induced activation and modulated the basal activity and cell surface translocation of xtMC3R and xtMC4R. In addition, the presence of two accessory proteins could convert xtMC3R and xtMC4R into ACTH-preferred receptors. These findings suggest that the presence of MRAPs exhibits fine control over the pharmacological activities of the neuronal MC3R and MC4R signaling in the Xenopus tropicalis, which is physiologically relevant with the complicated transition of feeding behaviors during their life history.

The capacity of digit tip regeneration observed both in rodents and humans establishes a foundation for promoting robust regeneration in mammals. However, stimulating regeneration at more proximal levels, such as the middle phalanges (P2) of the adult mouse, remains challenging. Having shown the effectiveness of transplantation of limb progenitor cells in stimulating limb regeneration in Xenopus, we are now applying the cell transplantation approach to the adult mouse. Here we report that both embryonic and induced pluripotent stem cell (iPSC)-derived limb progenitor-like cells can promote adult mouse P2 regeneration. We have established a simple and efficient protocol for deriving limb progenitor-like cells from mouse iPSCs. iPSCs are cultured as three-dimensional fibrin bodies, followed by treatment with combinations of Fgf8, CHIR99021, Purmorphamine and SB43542 during differentiation. These iPSC-derived limb progenitor-like cells resemble embryonic limb mesenchyme cells in their expression of limb-related genes. After transplantation, the limb progenitor-like cells can promote adult mouse P2 regeneration, as embryonic limb bud cells do. Our results provide a basis for further developing progenitor cell-based approaches for improving regeneration in the adult mouse limbs.

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.

The Xenopus tadpole is a favourable organism for regeneration research because it is suitable for a wide range of micromanipulative procedures and for a wide range of transgenic methods. Combination of these techniques enables genes to be activated or inhibited at specific times and in specific tissue types to a much higher degree than in any other organism capable of regeneration. Regenerating systems include the tail, the limb buds and the lens. The study of tail regeneration has shown that each tissue type supplies the cells for its own replacement: there is no detectable de-differentiation or metaplasia. Signalling systems needed for regeneration include the BMP and Notch signalling pathways, and perhaps also the Wnt and FGF pathways. The limb buds will regenerate completely at early stages, but not once they are fully differentiated. This provides a good opportunity to study the loss of regenerative ability using transgenic methods.

Bioelectric signals, particularly transmembrane voltage potentials (Vmem), play an important role in large-scale patterning during embryonic development. Endogenous bioelectric gradients across tissues function as instructive factors during eye, brain, and other morphogenetic processes. An important and still poorly-understood aspect is the control of cell behaviors by the voltage states of distant cell groups. Here, experimental alteration of endogenous Vmem was induced in Xenopus laevis embryos by misexpression of well-characterized ion channel mRNAs, a strategy often used to identify functional roles of Vmem gradients during embryonic development and regeneration. Immunofluorescence analysis (for activated caspase 3 and phosphor-histone H3P) on embryonic sections was used to characterize apoptosis and proliferation. Disrupting local bioelectric signals (within the developing neural tube region) increased caspase 3 and decreased H3P in the brain, resulting in brain mispatterning. Disrupting remote (ventral, non-neural region) bioelectric signals decreased caspase 3 and highly increased H3P within the brain, with normal brain patterning. Disrupting both the local and distant bioelectric signals produced antagonistic effects on caspase 3 and H3P. Thus, two components of bioelectric signals regulate apoptosis-proliferation balance within the developing brain and spinal cord: local (developing neural tube region) and distant (ventral non-neural region). Together, the local and long-range bioelectric signals create a binary control system capable of fine-tuning apoptosis and proliferation with the brain and spinal cord to achieve correct pattern and size control. Our data suggest a roadmap for utilizing bioelectric state as a diagnostic modality and convenient intervention parameter for birth defects and degenerative disease states of the CNS.

Biophysical forces play important roles throughout embryogenesis, but the roles of spatial differences in cellular resting potentials during large-scale brain morphogenesis remain unknown. Here, we implicate endogenous bioelectricity as an instructive factor during brain patterning inXenopus laevis. Early frog embryos exhibit a characteristic hyperpolarization of cells lining the neural tube; disruption of this spatial gradient of the transmembrane potential (Vmem) diminishes or eliminates the expression of early brain markers, and causes anatomical mispatterning of the brain, including absent or malformed regions. This effect is mediated by voltage-gated calcium signaling and gap-junctional communication. In addition to cell-autonomous effects, we show that hyperpolarization of transmembrane potential (Vmem) in ventral cells outside the brain induces upregulation of neural cell proliferation at long range. Misexpression of the constitutively active form ofNotch, a suppressor of neural induction, impairs the normal hyperpolarization pattern and neural patterning; forced hyperpolarization by misexpression of specific ion channels rescues brain defects induced by activated Notch signaling. Strikingly, hyperpolarizing posterior or ventral cells induces the production of ectopic neural tissue considerably outside the neural field. The hyperpolarization signal also synergizes with canonical reprogramming factors (POUandHB4), directing undifferentiated cells toward neural fatein vivo. These data identify a new functional role for bioelectric signaling in brain patterning, reveal interactions betweenVmemand key biochemical pathways (Notch and Ca2+signaling) as the molecular mechanism by which spatial differences ofVmemregulate organogenesis of the vertebrate brain, and suggest voltage modulation as a tractable strategy for intervention in certain classes of birth defects.

The tail of the Xenopus tadpole will regenerate completely after transection. Much of the mass of the regenerate is composed of skeletal muscle, but there has been some uncertainty about the source of the new myofibres. Here, we show that the growing tail contains many muscle satellite cells. They are active in DNA replication, whereas the myonuclei are not. As in mammals, the satellite cells express pax7. We show that a domain-swapped construct, pax7EnR, can antagonize pax7function. Transgenic tadpoles were prepared containing pax7EnR driven by a heat-inducible promoter. When induced, this reduces the proportion of satellite cells formed in the regenerate. A second amputation of the resulting tails yielded second regenerates containing notochord and spinal cord but little or no muscle. This shows that inhibition of pax7 action does not prevent differentiation of satellite cells to myofibres, but it does prevent their maintenance as a stem cell population.

Expression of myf-5, a key component of myogenic regulatory genes, expands into the ventral marginal zone during Xenopus gastrulation after the dorsal activation takes place. Little is known about how this dynamic expression pattern occurs. Here, we provide evidences to suggest that Activin/Nodal signals participate in the regulation of ventral expression of Xmyf-5 in gastrula embryos. Two Smad binding elements (SBEs) within the Xenopus myf-5 promoter can specifically interact with Smad4 protein. Furthermore, we demonstrate that the two SBEs are both indispensable for conferring responsiveness to Activin/Nodal signals and to ventral expression of myf-5 in Xenopus gastrula embryos.

For studies of vertebrate limb regeneration it is often desirable to visualize the regenerated skeleton, which is mostly cartilage, and also section the specimen for histological or immunohistochemical visualization of other tissues. However, the normal skeletal staining techniques are incompatible with immunohistochemistry. Here, we describe a contrast-based micro-computed tomography (microCT) method for direct and nondestructive observation of regenerated cartilage spikes in Xenopus frog limbs. In addition, we show that contrast based microCT imaging is compatible with immunohistochemistry protocols. This approach provides versatile and high contrast images of the cartilage allowing us to measure the regenerated skeletal structure in detail as well as carrying out the other types of analysis. It opens a wide range of potential microCT applications in research on vertebrate limb regeneration.

Background After amputation of the Xenopus tadpole tail, a functionally competent new tail is regenerated. It contains spinal cord, notochord and muscle, each of which has previously been shown to derive from the corresponding tissue in the stump. The regeneration of the neural crest derivatives has not previously been examined and is described in this paper. Results Labelling of the spinal cord by electroporation, or by orthotopic grafting of transgenic tissue expressing GFP, shows that no cells emigrate from the spinal cord in the course of regeneration. There is very limited regeneration of the spinal ganglia, but new neurons as well as fibre tracts do appear in the regenerated spinal cord and the regenerated tail also contains abundant peripheral innervation. The regenerated tail contains a normal density of melanophores. Cell labelling experiments show that melanophores do not arise from the spinal cord during regeneration, nor from the mesenchymal tissues of the skin, but they do arise by activation and proliferation of pre-existing melanophore precursors. If tails are prepared lacking melanophores, then the regenerates also lack them. Conclusion On regeneration there is no induction of a new neural crest similar to that seen in embryonic development. However there is some regeneration of neural crest derivatives. Abundant melanophores are regenerated from unpigmented precursors, and, although spinal ganglia are not regenerated, sufficient sensory systems are produced to enable essential functions to continue.

Myf-5, a member of the muscle regulatory factor family of transcription factors, plays an important role in the determination, development, and differentiation of the skeletal muscle. Factors that regulate the expression of myf-5 itself are not well understood. We show here that a T-box binding site in the Xenopus myf-5 promoter mediated the activation of myf-5 expression through specific interaction with nuclear proteins of gastrula embryos. The T-box binding site could be bound by and respond to T-box proteins. T-box genes could induce Xmyf-5. The results suggest that T-box proteins are involved in the specification of myogenic mesoderm and muscle development.