Self-renewal and differentiation of mammalian haematopoietic stem cells (HSCs) are controlled by a specialized microenvironment called the niche’. cortical zone (CZ), from a pool of multipotent progenitors called prohaemocytes present in the medullary zone (MZ)1. In addition, a group of signalling cells, termed posterior signalling centre (PSC), is clustered at the posterior end of the LG primary lobes2,3,4,5. Different signalling pathways have been shown to regulate the LG homoeostasis, that is, the balance between multipotent haemocyte progenitors Etoposide and differentiated blood cells6,7,8,9,10,11,12,13,14,15. Early analyses identified key roles of the transcription factor Collier (Col)/Knot and the morphogen Hedgehog (Hh), expressed in PSC cells. Increased haemocyte differentiation in the LGs mutant for either gene suggested that the PSC plays a role equivalent to the vertebrate haematopoietic niche in the bone marrow, in controlling the balance between progenitors and differentiating cells2,3. More recent studies revealed, however, that Col expression also defined a core population of progenitors in the LG MZ, and that massive differentiation of this Etoposide population occurred upon loss of Col expression in those cells16. The cell autonomous Col function required to maintain progenitors led to reinvestigating in more detail the PSC function under physiological conditions16,17. Data showed Etoposide that, Rabbit Polyclonal to STAT5A/B while not required for maintaining core progenitors16, the PSC controlled Etoposide the rate of haemocyte differentiation17, most likely by regulating the maturation of intermediate progenitors, a heterogeneous cell population in the third instar larval LG7,15,18,19. This role of the PSC is in accordance with previous studies showing that modifying the number of PSC cells altered the LG haemocyte differentiation3,6,14,20,21,22,23,24. Reinvestigating function in the LG also confirmed that the PSC plays an essential role in the mounting of a cellular immune response to wasp parasitism2,5,16,17,25. We previously found that bone morphogenetic protein/decapentaplegic (BMP/Dpp) signalling in PSC cells, controlled the number of these cells, via repression of the proto-oncogene mutants identified the Robo2 receptor as being expressed in the PSC, thereby raising the question of what role Slit/Robo signalling could play in these cells. Here we show that Slit/Robo signalling contributes to maintain the size, the morphology and the function of the PSC. Robo receptors are required in PSC cells to control both the proliferation rate and the clustering of these cells. The ligand Slit is expressed in the CT, that is, the vascular system, and might signal to Robos in the PSC. On the basis of our data, we propose that inter-organ communication between the CT and the PSC is required to preserve the morphology and function of the PSC. Results Abnormal PSC morphology in mutants Slit/Robo signalling is a key regulator of axon guidance, cell migration, adhesion and proliferation both in vertebrates and invertebrates26,27,28. Three Robo receptors and one Slit, the canonical Robo ligand, are encoded in the genome26,29,30. Examining the expression of Robo receptors by immunostaining with anti-Robo antibodies or by Etoposide looking at the expression of human influenza haemagglutinin (HA)-tagged endogenous alleles31, showed Robo1 was detected in the MZ, the CT and at low levels in the PSC, and Robo2 in PSC cells, crystal cells and in the CT (Fig. 1a; Supplementary Fig. 1aCf). Barely detectable levels if any of Robo 3 were present in PSC cells (Supplementary Fig. 1eCf). Thus, Robo1 and Robo2 are expressed in the PSC with at the highest level. To study the role of Robos in the LG, we first analysed a heterozygous context where one copy of robo2 was missing and observed an increase in PSC cell number (Fig. 1b,c). Furthermore, whereas PSC cells were.