Active hematopoietic sites in Drosophila Adult

Studies in the last decade have established Drosophila as the best invertebrate model to study hematopoiesis. Blood cell development in the fruitfly has been shown to have similarities to that of vertebrates both at the level of its origins and important signaling molecules necessary for their formation and differentiation (Evans et al., 2003). It was believed that active hematopoiesis is limited to embryonic and larval stages only and that the adult survives on the contribution of these earlier stages (Holz et al., 2003). However, adulthood is the most exploring phase of the fruit fly life, wherein chances of encountering different pathogens are comparatively high compared to larvae. We thus argued that the long lived hemocytes of earlier stages might not be sufficient to enable the fly to tide over diverse immune challenges.

We started off challenging the adult flies with bacteria. We noticed that post 24 hours infection the dorsal abdominal clusters were depleted of hemocytes and within another 48 hours they reverted back to wild type size. This made us wonder: how is the recovery achieved in these clusters? Is there any genesis of blood cells post infection within this cluster? Can these be the sites of new blood cell formation in the adult fruit fly? Our BrdU assay established that the otherwise quiescent plasmatocytes have entered into proliferation upon immune challenge indicating that they are not locked in senescence and can respond to insults.

For the past three years, we had been interested in exploring the contributions of embryo and larval stages to adulthood. Using the lineage tracing system we were trying to trace the lineages of hemocytes in the dorsal cluster of the adult Drosophila. In one such experiment, upon activation of the lineage tracing construct (Evans et al., 2009) with pan plasmatocyte driver hemolectin (hml) we were surprised to see several new plasmatocytes being born that lacked lineage tracing labeling but were actively expressing Hml indicating their genesis in adult.

We were excited to see a similar event also happening with crystal cells. This cell type is abundant in immature stages, but was reported to be absent in adult (Binggeli et al., 2014). Our analysis could detect them in a 5-day-old fly by Lozenge expression indicating the existence of a precursor from which they are derived. We decided to follow the crystal cell development in the dorsal abdominal cluster. Since activation of Notch (N) pathway precedes Lz expression in crystal cells (Duvic et al., 2002), a reporter of Notch pathway, Su(H) lacZ was employed for this purpose. Su(H) lacZ-positive cells are first seen in the cluster 2 post eclosion, whereas the expression of lz-GFP is observed only on 3 day post emergence. Interestingly, some of these lz- GFP-positive cells still have low levels of Su(H)lacZ expression. These results, therefore, clearly demonstrate the de novo origin of lz-GFP-positive crystal cells from Su(H)lacZ-positive cells within the cluster. These dorsal abdominal clusters are thus the active site for new blood cell formation and specification in adult fruit fly. This hemocyte aggregation is embedded in an intense network of extracellular matrix that facilitates the formation of the hub. On disturbing the plasmatocyte migration in earlier stages we found that larval hemocytes of do home into these hubs.

Deep seated, secluded from the rest of the abdominal cavity, this hematopoietic hub, enriched with Laminin A and collagen IV like protein, seems to be a simple version of the bone marrow. In vertebrates, the hematopoietic stem cells (HSCs) originate from hemangioblast and undergo maturation and expansion by an intricate developmental process that requires the involvement of the yolk sac, the aorta-gonad-mesonephros (AGM) region, the placenta and the fetal liver before finally colonizing into bone marrow (Mikkola and Orkin, 2006). Interesting, the progenitors that we detected in the fruit-fly hub arrive at the hub from the reserve population of hemocytes of the larval hematopoietic organ. This organ harbors blood cell progenitors originating from hemangioblasts that develop from a region analogous to the AGM of vertebrates (Mandal et al., 2004). Our investigation reveals that upon undergoing expansion within the lymph gland, some of these precursors actually home into the adult hematopoietic hub.

We believe that this simple version of bone marrow found through our investigation will help establish Drosophila adult hematopoiesis as a simpler yet genetically testable model to address questions related to blood stem cells as well as development , immunity, wound healing and aging (Ghosh et al., 2015).

Main paper:

Ghosh, S., Singh, A., Mandal, S., & Mandal, L. (2015). Active Hematopoietic Hubs in Drosophila Adults Generate Hemocytes and Contribute to Immune Response Developmental Cell, 33 (4), 478-488 DOI: 10.1016/j.devcel.2015.03.014

References

Binggeli, O., Neyen, C., Poidevin, M., & Lemaitre, B. (2014). Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila PLoS Pathogens, 10 (5) DOI: 10.1371/journal.ppat.1004067

Bossinger, B., Strasser, T., Janning, W., Klapper, R., & Holz, A. (2003). The two origins of hemocytes in Drosophila Development, 130 (20), 4955-4962 DOI: 10.1242/dev.00702

Duvic, B., Hoffmann, J., Meister, M., & Royet, J. (2002). Notch Signaling Controls Lineage Specification during Drosophila Larval Hematopoiesis Current Biology, 12 (22), 1923-1927 DOI: 10.1016/S0960-9822(02)01297-6

Evans, C., Olson, J., Ngo, K., Kim, E., Lee, N., Kuoy, E., Patananan, A., Sitz, D., Tran, P., Do, M., Yackle, K., Cespedes, A., Hartenstein, V., Call, G., & Banerjee, U. (2009). G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila Nature Methods, 6 (8), 603-605 DOI: 10.1038/nmeth.1356

Evans, C.J., Sinenko, S.A., Mandal, L., Martinez-Agosto, J.A., Hartenstein, V., & Banerjee, U. (2007). Genetic Dissection of Hematopoiesis Using Drosophila as a Model System Advances in Developmental Biology, 18, 259-299 DOI: 10.1016/S1574-3349(07)18011-X

Mikkola, H., & Orkin, S.H. (2006). The journey of developing hematopoietic stem cells Development, 133 (19), 3733-3744 DOI: 10.1242/dev.02568

Mandal, L., Banerjee, U., & Hartenstein, V. (2004). Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm Nature Genetics, 36 (9), 1019-1023 DOI: 10.1038/ng1404

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