Crayfish hematopoietic tissue as a model for stem cell development in arthropods

Arthropods, are suitable model animals to study the regulation of blood cell synthesis and differentiation of the innate immune system, since they lack the lymphocytes, and oxygen-carrying erythrocytes. In contrast to most insects, many crustaceans have a long life span and need to continuously synthesize blood cells. Crayfish hematopoiesis takes place in the hematopoietic tissue (HPT). The HPT of Pacifastacus leniusculus provides a simple model to study hematopoiesis because the tissue can be isolated and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. This tissue was earlier shown to be localized at the dorsal part of stomach. Here, we show that the HPT extends towards the anterior part the animal and link to the brain. Staining of HPT sections revealed that the most anterior of the tissue close to the brain contains higher percentage of cells with loose chromatin, whereas most of the cells in the posterior part have dark nuclear staining with condense chormatin. BrdU incorporation and immunostaining for phospho-histone H3 indicates that the actively proliferating cells occupy the anterior part of the tissue especially in the area close to the brain, proposed stem cell center (SCC). In contrast the more differentiated cells reside in the posterior part. Injection of LPS, which induced blood loss mimicking a bacterial infection, stimulated HPT cell proliferation especially in the anterior part of the tissue. High ROS level was found close to proliferating SCC and the brain, and laminarin-induced hemocyte loss caused induction of ROS level in SCC. This indicates the involvement of ROS in crayfish hematopoiesis. Isolated cells from SCC actively divide and form cell clusters whereas the cells from the remaining HPT from monolayer in in vitro culture. Collagen-I-matrix gel provided an appropriate environment for HPT cell culture and exhibited a suitable system to study HPT cell proliferation and differentiation indicating by induction of hemocyte marker transcripts. Being easily isolated and studied both in vitro and in vivo on stem cell proliferation as well as differentiation into mature hemocytes suggests that crayfish HPT provides an alternative simple model system to study hematopoiesis in arthropods. Moreover, the discovery of the astakine cytokines and antiapoptotic factor CHF offers an opportunity to explore the regulation of invertebrate hematopoiesis and its connection to the central nervous system as well as give new information on the evolution of different blood cell lineages.