The tail of the frog tadpole, comprising spinal cord, muscle, and notochord, regenerates following partial amputation. We show that, in Xenopus, this occurs throughout development, except for a "refractory period" between stages 45 and 47, when tails heal over without regeneration. Regeneration can be enabled during this refractory period by activation of either the BMP or Notch signaling pathways. Conversely, regeneration can be prevented during the later, regenerative, stages by inhibition of either pathway. BMP signaling will cause regeneration of all tissues, whereas Notch signaling activates regeneration of spinal cord and notochord, but not muscle. An activated form of Msx1 can promote regeneration in the same way as BMP signaling. Epistasis experiments suggest that BMP signaling is upstream of Notch signaling but exerts an independent effect on muscle regeneration. The results demonstrate that regenerative capability can be enabled by genetic modifications that reactivate specific components of the developmental program.

Bone morphogenetic protein (BMP) signalling is necessary for both the development of the tail bud and for tail regeneration in Xenopus laevis tadpoles. Using a stable transgenic line in which expression of the soluble BMP inhibitor noggin is under the control of the temperature inducible hsp70 promoter, we have investigated the timing of the requirement for BMP signalling during tail regeneration. If noggin expression is induced followed by partial amputation of the tail, then wound closure and the formation of the neural ampulla occur normally but outgrowth of the regeneration bud is inhibited. Furthermore, we show that BMP signalling is also necessary for limb bud regeneration, which occurs in Xenopus tadpoles prior to differentiation. When noggin expression is induced, limb bud regeneration fails at an early stage and a stump is formed. The situation appears similar to the tail, with formation of the limb bud blastema occurring but renewed outgrowth inhibited. The transcriptional repressor Msx1, a direct target of BMP signalling with known roles in vertebrate appendage regeneration, fails to be re-expressed in both tail and limb in the presence of noggin. DNA labelling studies show that proliferation in the notochord and spinal cord of the tail, and of the blastema in the limb bud, is significantly inhibited by noggin induction, suggesting that in the context of these regenerating appendages BMP is mainly required, directly or indirectly, as a mitogenic factor.

While Xenopus is a well-known model system for early vertebrate development, in recent years, it has also emerged as a leading model for regeneration research. As an anuran amphibian, Xenopus laevis can regenerate the larval tail and limb by means of the formation of a proliferating blastema, the lens of the eye by transdifferentiation of nearby tissues, and also exhibits a partial regeneration of the postmetamorphic froglet forelimb. With the availability of inducible transgenic techniques for Xenopus, recent experiments are beginning to address the functional role of genes in the process of regeneration. The use of soluble inhibitors has also been very successful in this model. Using the more traditional advantages of Xenopus, others are providing important lineage data on the origin of the cells that make up the tissues of the regenerate. Finally, transcriptome analyses of regenerating tissues seek to identify the genes and cellular processes that enable successful regeneration.

We have employed transgenic methods combined with embryonic grafting to analyse the mechanisms of regeneration in Xenopus tadpoles. The Xenopus tadpole tail contains a spinal cord, notochord and segmented muscles, and all tissues are replaced when the tail regenerates after amputation. We show that there is a refractory period of very low regenerative ability in the early tadpole stage. Tracing of cell lineage with the use of single tissue transgenic grafts labelled with green fluorescent protein (GFP) shows that there is no de-differentiation and no metaplasia during regeneration. The spinal cord, notochord and muscle all regenerate from the corresponding tissue in the stump; in the case of the muscle the satellite cells provide the material for regeneration. By using constitutive or dominant negative gene products, induced under the control of a heat shock promoter, we show that the bone morphogenetic protein (BMP) and Notch signalling pathways are both essential for regeneration. BMP is upstream of Notch and has an independent effect on regeneration of muscle. The Xenopus limb bud will regenerate completely at the early stages but regenerative ability falls during digit differentiation. We have developed a procedure for making tadpoles in which one hindlimb is transgenic and the remainder wild-type. This has been used to introduce various gene products expected to prolong the period of regenerative capacity, but none has so far been successful.

Background: Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. Here, we show that BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, we show that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall.

Determining the functional attributes of pancreatic transcription factors is essential to understand how the pancreas is specified distinct from other endodermal organs, such as liver, stomach and duodenum, and to direct the differentiation of other cell types into pancreas. Previously, we demonstrated that Pdx1-VP16 was sufficient to convert liver to pancreas. In this paper, we characterize the functional ability of another pancreatic transcription factor, Ptf1a, in promoting ectopic pancreatic fates at early stages throughout the endoderm and later during organogenesis. Using the transthyretin promoter to drive expression in the early liver region/bud of transgenic Xenopus tadpoles, we find that Ptf1a-VP16 is able to convert liver to pancreas. Overexpression of the unmodified Ptf1a on the other hand has no effect in liver but is able to convert stomach and duodenum to pancreas. When overexpressed at earlier embryonic stages throughout the endoderm, Ptf1a activity is similarly limited, whereas Ptf1a-VP16 has increased activity. Interestingly, in all instances we find that Ptf1a-VP16 is only capable of promoting acinar cell fates, whereas Ptf1a promotes both acinar and endocrine fates. Lastly, we demonstrate that, similar to mouse and zebrafish, Xenopus Ptf1a is essential for the initial specification of both endocrine and exocrine cells during normal pancreas development.

Homeobox genes encode positional information during primary and secondary axis formation during development. For this reason, the Hox genes have attracted attention in regeneration research as well. At early stages of regeneration, Hox genes have been implicated in wound healing and the dedifferentiation process and at later stages in the patterning of the blastema. We studied the expression of three Abdominal B-type Hox genes in Xenopus: XHoxc10, XHoxa13, and XHoxd13 during normal limb development and during regeneration of limbs and tails. We compared their expression with nonregenerating and with wounded limbs and tails, respectively. We show that the temporal and spatial control of these three Hox genes in blastemas differs from normal development. All three are specific to regeneration, XHoxc10 is up-regulated at the right time and at the site where cells dedifferentiate and undifferentiated cells are recruited, whereas XHoxa13 is reexpressed slightly later in regeneration, when the blastemal cells proliferate and remains on during patterning of the blastema. XHoxd13 is not expressed until relatively late and appears to be involved only in patterning of the blastema. Developmental Dynamics 226:349 -355, 2003.