Biomedical subjects

Elly M Tanaka

Publications and source records attributed to Elly M Tanaka.

12 recordsLinked to original sources

PubMed2006

A germline GFP transgenic axolotl and its use to track cell fate: dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration.

The development of transgenesis in axolotls is crucial for studying development and regeneration as it would allow for long-term cell fate tracing as well as gene expression analysis. We demonstrate here that plasmid injection into the one-cell stage axolotl embryo generates mosaic transgenic animals that display germline transmission of the transgene. The inclusion of SceI meganuclease in the injections (Thermes, V., Grabher, C., Ristoratore, F., Bourrat, F., Choulika, A., Wittbrodt, J., Joly, J.S., 2002. I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, 91-98) resulted in a higher percentage of F0 animals displaying strong expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of a transgene. Using this technique we have generated a germline transgenic animal expressing GFP ubiquitously in all tissues examined. We have used this animal to study cell fate in the dorsal fin during development. We have uncovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled hosts. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity.

Ambystoma↗

PubMed2006

Reversibility of the differentiated state: regeneration in amphibians.

In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, tail, eye lens, or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, dermis, and cartilage. In the case of multinucleated skeletal muscle fibers, cell cycle reentry into S-phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo.

Amphibians↗

PubMed2005

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins.

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.

Ambystoma mexicanum↗

PubMed2005

Proximodistal patterning during limb regeneration.

Regeneration is an ability that has been observed extensively throughout metazoan phylogeny. Amongst vertebrates, the urodele amphibians stand out for their exceptional capacity to regenerate body parts such as the limb. During this process, only the missing portion of the limb is precisely replaced--amputation in the upper arm results in regeneration of the entire limb, while amputation at the wrist produces a hand. Limb regeneration occurs through the formation of a local proliferative zone called the blastema. Here, we examine how proximodistal identity is established in the blastema. Using cell marking and transplantation experiments, we show that distal identities have already been established in the earliest stages of blastemas examined. Transplantation of cells into new environments is not sufficient to respecify cell identity. However, overexpression of the CD59, a cell surface molecule previously implicated in proximodistal identity during limb regeneration, causes distal blastema cells to translocate to a more proximal location and causes defects in the patterning of the distal elements of the regenerate. We suggest a model for the limb regeneration blastema where by 4 days post-amputation the blastema is already divided into distinct growth zones; the cells of each zone are already specified to give rise to upper arm, lower arm, and hand.

Ambystoma mexicanum↗

PubMed2005

Quantitative evaluation of morpholino-mediated protein knockdown of GFP, MSX1, and PAX7 during tail regeneration in Ambystoma mexicanum.

Vertebrate regeneration is a fascinating but poorly understood biological phenomena. Urodele amphibians such as Ambystoma mexicanum (the axolotl) can functionally regenerate complex body structures such as the limb and tail, including the spinal cord, throughout life. So far, molecular studies on regeneration have been limited due to the paucity of tools for knocking-down gene and protein function. In this article, we quantitatively assessed the ability of morpholinos to specifically down-regulate protein expression in both cultured urodele cells and in vivo. We focused on the down-regulation of green fluorescent protein and two axolotl proteins, MSX1 and PAX7. Our data show that the expression of these proteins can be efficiently reduced by morpholinos. MSX1 has been hypothesized to be involved in muscle dedifferentiation based on experiments using cultured myotubes. Our studies in in vivo muscle fibers so far have shown no influence of overexpressing or down-regulating MSX1 on the dedifferentiation process.

Ambystoma mexicanum↗

PubMed2005

Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration.

Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord, cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. We investigated this phenomenon on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicated that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells was severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments revealed that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema, albeit with a different topography to mediate growth and patterning of tail tissues during regeneration.

Ambystoma↗

PubMed2004

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

BACKGROUND: The 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. RESULTS: Two 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. CONCLUSIONS: Our 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.

Ambystoma↗

PubMed2004

Plasticity and reprogramming of differentiated cells in amphibian regeneration: partial purification of a serum factor that triggers cell cycle re-entry in differentiated muscle cells.

The reversal of cellular differentiation to form proliferating progenitor cells is a critical aspect of regenerative ability in the urodele amphibians. This process has been studied using skeletal muscle during limb or tail regeneration, or dorsal iris epithelium during lens regeneration. An unknown activity in serum triggers cell cycle re-entry from the differentiated state. Here we describe the biochemical properties and fractionation of this serum factor. The factor is a glycoprotein that associates with large molecular weight complexes. The purification and molecular identification of the serum factor represents an important avenue in understanding regenerative ability and dedifferentiation capacity on a molecular basis.

Animals↗

PubMed2003

Regeneration: if they can do it, why can't we?

The therapeutic potential of stem cells and nuclear cloning has led to renewed interest in classical models of regeneration. This longstanding problem is undergoing a renaissance spurred by the availability of new techniques that finally allow analysis on the cellular and molecular level.

Animals↗

PubMed2003

Cell differentiation and cell fate during urodele tail and limb regeneration.

One of the most striking natural examples of adult tissue plasticity in vertebrates is limb and tail regeneration in urodele amphibians. In this setting, amputation triggers the destabilization of cell differentiation and the production of progenitor cells that extensively proliferate and pattern themselves to recreate a perfect replica of the missing part. A precise understanding of which cells dedifferentiate and how plastic they become has recently begun to emerge. Furthermore, information on which developmental gene programs are activated upon injury is becoming better understood. These studies indicate that, upon injury, an unusual cohort of genes are co-expressed. The future challenge will be to link the systems for studying dedifferentiation with activation of gene expression to understand on a molecular level how cells are 'pushed backward' to regenerate a complex structure such as a limb or tail.

Animals↗

PubMed2002

Ectoderm to mesoderm lineage switching during axolotl tail regeneration.

Foreign environments may induce adult stem cells to switch lineages and populate multiple tissue types, but whether this mechanism is used for tissue repair remains uncertain. Urodele amphibians can regenerate fully functional, multitissue structures including the limb and tail. To determine whether lineage switching is an integral feature of this regeneration, we followed individual spinal cord cells live during tail regeneration in the axolotl. Spinal cord cells frequently migrate into surrounding tissue to form regenerating muscle and cartilage. Thus, in axolotls, cells switch lineage during a real example of regeneration.

Ambystoma↗

PubMed2002

Mechanisms of muscle dedifferentiation during regeneration.

For many years people have known that amphibians have an amazing ability to regenerate lost body parts. In contrast humans have limited regeneration capacity and even simple wound healing results in scarring. Despite more than a century of scientific inquiry, this remarkable phenomenon remains poorly understood. Recent research has begun to provide insight into how this unique process that is now fully accepted to occur via the reversal of cell differentiation is executed at the molecular level. As more and more is known about regeneration and dedifferentiation we can begin to address the question: if given the right signals could mammals also regenerate body structures?

Animals↗