Protection from UV-Light Is an Evolutionarily Conserved Feature of the Hematopoietic Niche
Authors: Kapp Friedrich G , Perlin Julie R , Trompouki Eirini , Niemeyer Charlotte M , Driever Wolfgang , Zon Leonard I. 
Published: October 2019

Abstract
Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow cavity in all mammals, but other locations are used in non-mammalian animals, such as the kidney marrow in zebrafish. The niche location is thought to be selected based on unique advantages for HSPC function and protection in each organism. Studying the zebrafish HSPC niches during development and adulthood, we observed that melanocytes occupy the location above the kidney prior to the arrival of HSPCs. To elucidate whether these particular melanocytes play a role for HSPCs, we compared HSPCs in wildtype and nacre-mutant embryos (that lack all melanocytes) by cmyb in situ at different time-points. There were no obvious differences, suggesting that melanocytes are dispensable for steady state hematopoiesis. Since the position of the melanocytes above the HSPCs was very consistent in different strains and at various ages, we asked whether the melanocytes protect the HSPC niche from UV light. Indeed, after irradiation with UV light, levels of UV-induced DNA damage were approximately 50% higher in HSPCs isolated from kidneys of unpigmented embryos compared to their pigmented siblings as assessed by immunocytochemistry with a cyclobutane pyrimidine dimer antibody (TDM-2). In addition to acquiring more DNA damage, the unpigmented embryos also showed a significantly more pronounced decrease of HSPCs as assessed by cmyb in situ: 55% of control embryos had high cmyb staining. Upon irradiation 44% of irradiated pigmented embryos retained high c-myb staining compared to only 28% of irradiated unpigmented embryos (total embryos analyzed: n= 29, 27 and 36 respectively; p=0.01 between the latter two groups). The reduction of HSPCs could not be rescued in unpigmented p53-mutant embryos, indicating a p53-independent mechanism of HSPC loss after UV irradiation. The protective effect of melanocytes could either be due to a physical shielding of the HSPCs or due to a paracrine mechanism, such as secretion of cytokines or uptake of reactive oxygen species. The paracrine mechanism could be dependent on melanin. To address this question, we focused the path of light at a different angle in which the melanocytes would not be able to block the UV light from reaching the kidney marrow. This abrogated the protective effect of pigment containing melanocytes and caused a reduction of HSPCs by cmyb in situ to the levels of unpigmented siblings: pigmented embryos irradiated at an angle now also had high cmyb staining in only 28% of the embryos (n= 25). This suggests that the melanocytes only provide shade for the HSPCs. We then asked if this protective pigmentation pattern is specific to zebrafish and traced the origin of this trait by comparative histology. We found that other teleost fish but also evolutionarily older fish such as sturgeon and polypterus have melanocytes covering the kidney marrow. Even in lamprey, an ancient jawless vertebrate, the HSPC niche (located in the supraspinal organ) is covered by melanocytes, which allows us to date the evolutionary origin of UV protection back to at least 500 million years. We performed histological studies in frogs and found that the HSPCs reside under a melanocyte shield in the tadpole stage, while it is known that in adult frogs the HSPCs move into the bone marrow cavity. Considering that all terrestrial animals such as reptiles, birds and mammals host hematopoiesis in the bone marrow, we suggest that during the transition from aquatic to terrestrial life, the HSPCs were driven out of their melanocyte covered niche due to the high level of irradiation and relocated into a more protective niche, the bone marrow.

Disclosures
Zon: Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.