Hematopoietic ontogeny in the axolotl

In this issue of Blood, Lopez et al undertake the heroic task of characterizing the blood-forming system of the axolotl (Ambystoma mexicanum), an aquatic salamander that provides an excellent model for tissue regeneration and scar-free wound healing.¹  Commonly referred to as the “Mexican walking fish,” axolotls are not fish at all, but rather neotenic salamanders that retain many larval traits throughout their lifespan because they do not undergo a typical juvenile to adult metamorphosis. This retention of larval traits is associated with the profound ability of the axolotl to regenerate many of its tissues, including limbs, spinal cord, heart, and even parts of its brain.^2-6 

The regenerative mechanisms of the axolotl have been reasonably well studied, and the involvement of specific hematopoietic lineages in these processes has been documented.^7,8  However, there remains a lack of knowledge regarding the full spectrum of hematopoietic cells present, techniques to mark these cells, and assays to test their functions. Although the axolotl generates blood cell lineages similar to other vertebrates,^8,9  where, when, and how these cells arise have remained enigmatic.

In this study, Lopez et al present a first characterization of the embryonic and adult hematopoietic systems of these animals (see figure for an overview). They show that, like blood cells of mammals and zebrafish, axolotl blood cells can be characterized and isolated by fluorescence activated cell sorting based on their size and granularity. Morphological analyses of these cells suggest that the major blood cell lineages found in other vertebrates are present, including lymphocytes, monocytes, macrophages, mast cells, and neutrophils. Importantly, they also present assays to test the function of hematopoietic stem and progenitor cells (HSPCs), which they use to investigate the sites of hematopoiesis in the adult animal. Interestingly, unlike mammals and teleosts, there exist 2 sites of hematopoiesis in the adult axolotl. By developing colony-forming unit (CFU) assays to reveal erythroid and multilineage potential, the authors demonstrate that both the liver and spleen harbor robust CFU activity. In contrast, the bone marrow, thymus, and kidney generated no hematopoietic colonies. The authors subsequently developed transplantation assays to test each tissue for long-term repopulating hematopoietic stem cells (HSCs). Consistent with the CFU results, only the liver and spleen contained HSCs, as evidenced by long-term, donor-derived hematopoiesis in irradiated recipient animals. Importantly, the authors identify an array of lineage-specific antibodies that can be used to demonstrate multilineage reconstitution. Finally, they show convincing evidence that ablation of the liver or spleen by targeted irradiation led to anemia and subsequent death of the animals, supporting the key roles of each tissue in maintaining the hematopoietic program.

After characterizing the hematopoietic system in the adult axolotl, the authors examined HSPC ontogeny during development. Transplantation of green fluorescent protein–positive (GFP⁺) cells from transgenic donor embryos demonstrated that the blood islands lacked HSC activity. Interestingly, the first organ to harbor HSCs capable of long-term engraftment was the embryonic liver, followed by the embryonic spleen.

These studies open the door for further in-depth analyses of the axolotl hematopoietic system. By using GFP⁺ transgenic donors, hematopoietic cells can be easily visualized in adults, which is advantageous for future studies characterizing the role of specific cell types in wound repair and regeneration, as well as visualizing homing and engraftment. Additionally, the authors show that HSPC populations are roughly 1000-fold enriched in the lymphoblastic population of the spleen, positioning this fraction for further prospective isolation approaches. In addition, their finding that HSPCs reside in the peripheral layer of the liver indicates that the axolotl liver and spleen likely provide different hematopoietic niches for different functions. Comparison of the signaling processes occurring between these 2 sites, as well as among other niches in different vertebrate animals, may provide insight into the conserved core network of HSC support molecules. Finally, the axolotl is likely an excellent system to understand the role of hematopoietic cells not only in the regeneration of limbs, spinal cord, and other organs, but also in the regeneration of the hematopoietic system itself. For example, as mammals age, they have less ability to robustly generate lymphoid cells, with HSCs becoming skewed toward myeloid outputs. It would be interesting to investigate if this trend exists in the neotenic axolotl, which in the adult form retains juvenile traits. Overall, this pioneering work in the axolotl now provides another excellent model system in which to compare and contrast the evolution of vertebrate hematopoiesis.