David M. Gardiner

Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration.

BackgroundMicroarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration.ResultsConsiderable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences.ConclusionMany new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

Retinoic acid, local cell-cell interactions, and pattern formation in vertebrate limbs

Retinoic acid (RA), a derivative of vitamin A, has remarkable effects on developing and regenerating limbs. These effects include teratogenesis, arising from RAs ability to inhibit growth and pattern formation. They also include pattern duplication, arising as a result of the stimulation of additional growth and pattern formation. In this review we present evidence that the diverse effects of RA are consistent with a singular, underlying explanation. We propose that in all cases exogenously applied RA causes the positional information of pattern formation-competent cells to be reset to a value that is posterior-ventral-proximal with respect to the limb. The diversity of outcomes can be seen as a product of the mode of application of exogenous RA (global versus local) coupled with the unifying concept that growth and pattern formation in both limb development and limb regeneration are controlled by local cell-cell interactions, as formulated in the polar coordinate model. We explore the possibility that the major role of endogenous RA in limb development is in the establishment of the limb field rather than as a diffusible morphogen that specifies graded positional information across the limb as previously proposed. Finally, we interpret the results of the recent finding that RA can turn tail regenerates into limbs, as evidence that intercalary interactions may also be involved in the formation of the primary body axis.

Expression of Msx‐2 during development, regeneration, and wound healing in axolotl limbs

Msx genes are transcription factors that are expressed during embryogenesis of developing appendages in regions of epithelial-mesenchymal interactions. Various lines of evidence indicate that these genes function to maintain embryonic tissues in an undifferentiated, proliferative state. We have identified the axolotl homolog of Msx-2, and investigated its expression during limb development, limb regeneration, and wound healing. As in limb buds of higher vertebrates, axolotl Msx-2 is expressed in the apical epidermis and mesenchyme; however, its expression domain is more extensive, reflecting the broader region of the apical epidermal cap in amphibians. Msx-2 expression is downregulated at late stages of limb development, but is reexpressed within one hour after limb amputation. Msx-2 is also reexpressed during wound healing, and may be essential in the early stages of initiation of the limb regeneration cascade.

Expression of Mmp-9 and Related Matrix Metalloproteinase Genes During Axolotl Limb Regeneration

One of the earliest events in limb regeneration is the extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are a family of matrix degrading enzymes that have been identified in both normal and disease states. Using RT‐PCR and cDNA library screening, we have isolated sequences homologous to four different Mmp genes. The spatial and temporal expression of one of these, Mmp‐9, has been analyzed during axolotl limb regeneration. Northern blot analysis identifies a 3.8 kb transcript that is abundantly expressed during regeneration, and whole‐mount in situ hybridization has uncovered an unusual bi‐phasic expression pattern. The first phase begins at 2 hours after amputation, and expression is confined to the healed wound epithelium. This phase continues for 2 days, showing peak expression at 14 hours after amputation. This early phase may be needed to retard reformation of the basal lamina of the epidermis, and thereby facilitate the epidermal‐mesenchymal interactions required for successful regeneration. The second phase begins a few days later when a small blastema has formed. During this phase, expression is in the mesenchyme, localized to cells around the tips of the cut skeletal elements. This expression is maintained through several stages until redifferentiation begins. The timing and position of the second phase of expression is consistent with a role for Mmp‐9 in the removal of damaged cartilage matrix. We have also discovered that the time of onset of Mmp‐9 expression is sensitive to denervation, which causes a delay of several hours. Finally, retinoids, known for their dramatic effects on the pattern of regenerating limbs, can cause a down regulation of Mmp‐9 expression. Dev Dyn 1999;216:2–9.

Neurotrophic regulation of epidermal dedifferentiation during wound healing and limb regeneration in the axolotl (Ambystoma mexicanum)

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. In the present study, we have utilized the ALM to identity the buttonhead-like zinc-finger transcription factor, Sp9, as being involved in the formation of the regeneration epithelium. Sp9 expression is induced in basal keratinocytes of the apical blastema epithelium in a pattern that is comparable to its expression in developing limb buds, and it thus is an important marker for dedifferentiation of the epidermis. Induction of Sp9 expression is nerve-dependent, and we have identified KGF as an endogenous nerve factor that induces expression of Sp9 in the regeneration epithelium.

The migration of dermal cells during blastema formation in axolotls

Using the diploid/triploid cell marker in the axolotl (Ambystoma mexicanum) we have examined the movement of cells from the dermis into the early limb blastema. Cells of dermal origin begin to migrate beneath the wound epithelium at about 5 days postamputation, and by 10 days they are widely distributed across the amputation surface. By 15 days, a dense accumulation of blastema cells is present beneath the apical cap, and these cells are preferentially oriented in a circumferential direction. These results are discussed in relation to previous studies showing that the progeny of dermal cells become widely distributed during regeneration, and that cells of dermal origin are a major source of blastema cells. The results are also discussed in relation to ideas about how growth and patterning of the new appendage occur.

From biomedicine to natural history research: EST resources for ambystomatid salamanders

BackgroundEstablishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories.ResultsApproximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species.ConclusionsOur study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.

Sonic Hedgehog (shh) expression in developing and regenerating axolotl limbs

Sonic hedgehog (shh) expression is detectable in the posterior mesenchyme of many developing vertebrate limbs. We have isolated an RT-PCR fragment from the axolotl, Ambystoma mexicanum, that has high identity to other vertebrate shh genes. We describe the localization of this transcript during development and regeneration and in response to tissue grafts and retinoic acid (RA) exposure in the axolotl. Even though axolotl digits show a reversed polarity of differentiation (anterior [A] to posterior [P]) when compared to other tetrapods (P to A), shh is nevertheless expressed on the posterior margin of developing and regenerating limb buds. When A cells are grafted adjacent to P cells, an ectopic domain of shh is induced. Exposure to retinoic acid (RA), a molecule known to alter pattern in all three limb axes in urodeles, results in ectopic expression of shh in anterior cells of the regeneration blastema. Prior to this induced expression in response to RA, there is an earlier response by the endogenous domain of shh, which is downregulated within the first few hours of exposure.

The molecular basis of amphibian limb regeneration: integrating the old with the new.

Is regeneration close to revealing its secrets? Rapid advances in technology and genomic information, coupled with several useful models to dissect regeneration, suggest that we soon may be in a position to encourage regeneration and enhanced repair processes in humans.

Environmentally induced limb malformations in mink frogs (Rana septentrionalis)

In recent years, there has been an increase in the incidence of frog deformities throughout many of the northern states of North America. The most readily noticed malformations involve the hindlimbs of peri-metamorphic animals. We have analyzed skeletal preparations of metamorphosing mink frogs (Rana septentrionalis) collected from a site in Minnesota, in order to develop a better understanding of the possible causes. In this paper we describe the categories of abnormalities found at this site. The spectrum of deformities includes missing limbs, truncated limbs, extra limbs (including extra pelvic girdles), and skin webbings. We also describe a newly recognized malformation of the proximal-distal limb axis, a bony triangle. In this abnormality, the proximal and distal ends of the bone are adjacent to one another forming the base of a triangle. The shaft of the bone is bent double and protrudes laterally, the midpoint of the bone forming the apex of the triangle. In this paper we consider several recently proposed explanations for the recent outbreak of amphibian deformities. Based on our analysis, we conclude that the spectrum of abnormalities seen in these frogs is remarkably similar to the range of abnormalities that has been reported as a result of exposure of developing vertebrates to exogenous retinoids. Given the potential implications of this possibility for the welfare of humans as well as wildlife, further studies are needed to determine whether environmental retinoids are responsible for the frog deformities at the site we have examined.