Hans H. Epperlein

Cells keep a memory of their tissue origin during axolotl limb regeneration.

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

A clonal analysis of neural progenitors during axolotl spinal cord regeneration reveals evidence for both spatially restricted and multipotent progenitors

Complete regeneration of the spinal cord occurs after tail regeneration in urodele amphibians such as the axolotl. Little is known about how neural progenitor cells are recruited from the mature tail, how they populate the regenerating spinal cord, and whether the neural progenitor cells are multipotent. To address these issues we used three types of cell fate mapping. By grafting green fluorescent protein-positive (GFP+) spinal cord we show that a 500 μm region adjacent to the amputation plane generates the neural progenitors for regeneration. We further tracked single nuclear-GFP-labeled cells as they proliferated during regeneration, observing their spatial distribution, and ultimately their expression of the progenitor markers PAX7 and PAX6. Most progenitors generate descendents that expand along the anterior/posterior (A/P) axis, but remain close to the dorsal/ventral (D/V) location of the parent. A minority of clones spanned multiple D/V domains, taking up differing molecular identities, indicating that cells can execute multipotency in vivo. In parallel experiments, bulk labeling of dorsally or ventrally restricted progenitor cells revealed that ventral cells at the distal end of the regenerating spinal cord switch to dorsal cell fates. Analysis of PAX7 and PAX6 expression along the regenerating spinal cord indicated that these markers are expressed in dorsal and lateral domains all along the spinal cord except at the distal terminus. These results suggest that neural progenitor identity is destabilized or altered in the terminal vesicle region, from which clear migration of cells into the surrounding blastema is also observed.

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

BackgroundThe ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum.ResultsTwo cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians.ConclusionsOur analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online.

BMP-4 and Noggin signaling modulate dorsal fin and somite development in the axolotl trunk.

BMP‐4, a member of the TGF‐beta superfamily of growth factors, is involved in various developmental processes. We investigated the effects of BMP‐4 and its antagonist Noggin on axolotl trunk development. Implantation of BMP‐4‐coated microbeads caused inhibition of muscle and dorsal fin formation in the vicinity of the microbeads. At some distance, myotomes developed with reduced height but increased width, which was accompanied by increased cell proliferation. These effects could be modulated by co‐implanting Noggin‐coated beads. Immunostaining of Pax7 further revealed that although the dermomyotome was absent in the vicinity of BMP‐4‐coated beads, at some distance from them, it was thicker than in controls, indicating that moderate amounts of BMP‐4 stimulate this layer of undifferentiated cells. In contrast, Noggin generally inhibited the dermomyotome, possibly indicating premature differentiation of dermomyotome cells. We conclude that BMP‐4 and Noggin are involved in the regulation of cell proliferation and differentiation during somite development. Developmental Dynamics 236:2464–2474, 2007.

Axolotl (Ambystoma mexicanum) embryonic transplantation methods.

Cold Spring Harb Protoc Eugen Nacu, Dunja Knapp, Elly M. Tanaka and Hans H. Epperlein ) Embryonic Transplantation Methods Ambystoma mexicanum Axolotl ( Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (873 articles) Laboratory Organisms, general (190 articles) Imaging Development (283 articles) Emerging Model Organisms (563 articles) Developmental Biology (988 articles) Cell Biology, general

Axolotls with an under- or oversupply of neural crest can regulate the sizes of their dorsal root ganglia to normal levels

How animals adjust the size of their organs is a fundamental, enduring question in biology. Here we manipulate the amount of neural crest (NC) precursors for the dorsal root ganglia (DRG) in axolotl. We produce embryos with an under- or over-supply of pre-migratory NC in order to find out if DRG can regulate their sizes during development. Axolotl embryos are perfectly suitable for this research. Firstly, they are optimal for microsurgical manipulations and tissue repair. Secondly, they possess, unlike most other vertebrates, only one neural crest string located on top of the neural tube. This condition and position enables NC cells to migrate to either side of the embryo and participate in the regulation of NC cell distribution. We show that size compensation of DRG in axolotl occurs in 2 cm juveniles after undersupply of NC (up-regulation) and in 5 cm juveniles after oversupply of NC (down-regulation). The size of DRG is likely to be regulated locally within the DRG and not via adaptations of the pre-migratory NC or during NC cell migration. Ipsi- and contralateral NC cell migration occurs both in embryos with one and two neural folds, and contralateral migration of NC is the only source for contralateral DRG formation in embryos with only one neural fold. Compensatory size increase is accompanied by an increase in cell division of a DRG precursor pool (PCNA+/SOX2-), rather than by DRG neurons or glial cells. During compensatory size decrease, increased apoptosis and reduced proliferation of DRG cells are observed.

19-P005 Size regulation of dorsal root ganglia in developing axolotls with an under- or oversupply of neural crest material

to correspond to the plasticity of the brain. In zebrafish many distinct constitutive proliferation zones have been described along the whole anterior–posterior axis of the brain [Grandel et al. Dev. Biol. 295 (2006) 263–277]. The cns of teleosts has an enormous capacity to regenerate after brain incisions or removal of whole brain parts, as well as after transection of the spinal cord. Neural regeneration has been demonstrated in several teleost species suggesting that it is a common feature of teleost fishes in general [Kaslin et al. Philos. Trans. R Soc. Lond. B Biol. Sci. 363 (2008) 101–122]. To investigate regenerative processes in the cns of adult zebrafish we have established a stab-wound lesion paradigm in the telencephalon. The temporal course of regenerative events was analyzed. After lesion a marked increase in proliferation was detected in the constitutive proliferation zones, as well as ectopic proliferation proximal to the lesion. We are currently analyzing the identity and proliferative properties of cells contributing to regeneration using BrdU to specifically label cycling cells combined with immunohistological detection of neuronal and glial marker proteins.

09-P046 Long-term fate mapping of neural crest and mesoderm in the shoulder girdle of the axolotl (Ambystoma mexicanum)

myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Lossand gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally we use live imaging to show that cebpa expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus C/EPB alpha is the first known single factor capable of initiating an entire myelopoeisis pathway in pluripotent cells in the embryo.

09-P101 Sources of striated muscle in the tail of developing axolotls

move inwards on the surface of, yet solid, oral endoderm. On the basis of this movement, ectoderm cells become the basal layer, whereas endoderm cells become the apical layer of the definite oral epithelium. As a direct consequence, the endoderm cells are observed even at an outer lip surface outside of mouth, as well as significantly contribute to tooth anlagen. This alternative mode of axolotl mouth development clearly demonstrates a great deal of flexibility in the formation of oral region in vertebrates.

19-P015 Plasticity of cells during axolotl limb regeneration

ase, for which we have identified several potential candidate genes. Inactivating H,K-ATPase activity with the highly potent and specific inhibitor, SCH-28080 (SCH), is sufficient to block regeneration of visible anterior structures in cut fragments. Marker analysis confirms a lack of anterior development, but without any duplication of posterior patterning, suggesting H,K-ATPase activity plays an important role in the anterior patterning of new tissues. Physiologically, membrane-voltage-sensitive dyes reveal that SCH treatment hyperpolarizes regenerating fragments. Our investigations have shown treatment with SCH blocks anterior polarity only in posterior fragments, and that the presence of the cephalic ganglia (brain) in any fragment is sufficient to rescue head development. Interestingly, there must be sufficient length along the A/P axis of a fragment for SCH to be effective, because fragments with significantly small A/P distances do not respond to SCH and are indistinguishable from controls. These data suggest that non-local A/P polarity information may be transmitted through ion flows along the A/P axis, providing information about the original A/P patterning of existing tissues that in turn regulates blastema morphology.