1412 Veronica Basso et al. Eur. J. Immunol. 2011. 41: 1410–1419 Figure 1. Rac1N/Rac3KO double-deﬁcient mice exhibit reduced size and cellullarity of primary and secondary lymphoid organs. WT, Rac1N, Rac3KO, and Rac1N/Rac3KO mice were sacriﬁced at P13. (A) Body weight of P13 mice. (B and D) Representative (B) thymi and (D) spleens from Rac3KO and Rac1N/Rac3KO. (C and E) Thymi and spleens were explanted and reduced to single-cell suspensions. Total numbers of viable cells in the (C) thymus and (E) spleen were obtained by (A, C, and E) Trypan blue counts. Each symbol represents data from an individual mouse; horizontal bar represents the mean. po0.005; po0.001 by one-way ANOVA with Bonferroni correction. both Rac3KO and Rac1N/Rac3KO (Supporting Information Fig. 3). Although IL-7, a nonredundant trophic factor for immune cell development [15], was expressed in both Rac3KO and Rac1N/ Rac3KO thymi (Supporting Information Fig. 4A), variability among independent experiments failed to overrule a possible defect along this pathway in mutant mice. Against this possibility, DN thymocytes proliferated to comparable extents in short BrdU- pulse experiments in vivo (Supporting Information Fig. 4C). In some of the Rac1N/Rac3KO sections the cortical region appeared to contain fewer CD31 precursors, which instead appeared better represented within the more densely populated medullary region. Also, Foxp31 cells, identifying T-regulatory cells exclusively present in the thymic medulla, were better represented in Rac1N/Rac3KO when compared with Rac3KO mice (Supporting Information Fig. 3). Histological analysis of the spleen revealed an altered red/white pulp ratio in Rac1N/Rac3KO, possibly reﬂecting the lower numbers of T and B cells, major constituents of the white pulp (Supporting Information Fig. 5A and B). In addition to the thymus and the spleen, the size (data not shown) and weight (Supporting Information Fig. 6A) of other organs including kidney, heart, and liver were also signiﬁcantly & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim smaller in P13 double KO animals when compared with Rac3KO littermates. In contrast, the size and weight of the brain [14] and the lung (Supporting Information Fig. 6A) were comparable in Rac3KO and Rac1N/Rac3KO mice. In all cases, the histological appearance of non-lymphoid tissues of Rac1N/Rac3KO mice appeared normal (Supporting Information Fig. 6B). Together these data indicate that the manifestation of neurological symptoms inﬂuences the development/growth of several vital organs to different extents, severely hindering immune-competence in Rac1N/Rac3KO double-mutant mice. Neuronal depletion of Rac1 and Rac3 is responsible for the effects observed on lymphoid organs Rac1N/Rac3KO mice carry the full deletion of the neural-speciﬁc gene for Rac3 (Rac3KO), and the conditional neuronal deletion of the gene for Rac1 by the Synapsin-Cre transgene (Rac1N). We reasoned that the developmental defects observed in the spleen and thymus of Rac1N/Rac3KO mutant mice was not attributable to a defect of endogenous Rac function in these organs. To better www.eji-journal.eu