Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration

Abstract

Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration.

Keywords: Axolotl; C59; Chemical screening; Regeneration; Wnt.

Figure 1. The axolotl tail amputation assay uses developmental stage 42 (Bordzilovskaya et al. 1989) embryos that are approximately 1 cM in total body length

The proportional increase in body size is determined 7 days post-amputation from images that document total body length.

Figure 2. Plot showing the average increase in body length after amputating approximately 2 mm from the distal tail tip of an axolotl embryo

The error bars are standard deviations of the mean.

Figure 3. Panel A shows the average proportional increase in body size at day 7 for embryos that were administered tail amputations and reared with or without (controls) chemical inhibitors of Wnt (C59), Fgf (BGJ398), and Tgf (SB505124) signaling pathways

The error bars are standard deviations of the mean. The difference between control and treatment mean is significant for each chemical treatment (Students 2-tailed T test, p < 0.001). Panel B shows representative pictures of the effects of the three chemicals on embryos at day 7.

Figure 4. Design of the microarray experiment and tissue sampling strategy

The red dotted lines show the amount of tissue collected at Day 0 and Day 7. During the time course, samples from control embryos are progressively enriched for regenerating tissue relative to embryos reared in C59

Figure 5

The number of genes that were identified as statistically significant and > 1.5 fold differently expressed between control and C59-treated embryos at each time tissues were collected for RNA isolation.

Figure 6. Expression profiles for genes that diverged significantly in expression between control and C59-treated embryos at 24 HPA (A), 48 HPA (B), 72 HPA (C), and 120 and168 HPA (D)

The numbers on the y-axes of each plot show the range of log (2) expression values. The error bars are standard deviations of the mean.