For example, DN1 cells migrate to the thymic capsule in response to CXCR4 ligand (CXCL12/SDF1) produced by the thymic cortex (Plotkinet al., 2003). bidirectional signaling between developing thymocytes and thymic epithelial cells (TEC) along with cues from chemokine gradients (Anderson and Jenkinson, 2001). The thymic capsule and TEC precursors originate from a single third pharyngeal pouch after its disconnection from the third cleft ectoderm (Rodewald, 2008). Thymic organogenesis begins upon thymocyte precursor migration to the thymic anlage. As incoming thymocytes pass through the initial stages of differentiation, they are first classified as double unfavorable (DN) by the lack of expression of CD4 and CD8 and further characterized by the expression of CD44 and CD25. These immature DN thymocytes induce the development of thymic corticomedullary structures by affecting the development and proliferation of cortical epithelial cells (cTEC) or medullary epithelial cells (mTEC) ORY-1001(trans) (Rossiet al., 2006). Cortical structure is usually disorganized in the thymus ofhCD3tg mice where T cell development is blocked at the CDD44+CD25DN1 stage but cortical structure is well organized in RAG2/mice where T cell development is blocked at ORY-1001(trans) the CD44CD25+DN3 stage, suggesting that CD4CD8thymocytes expressing CD44 and CD25 (DN2) are ORY-1001(trans) required for well-organized corticomedullary structure (Klug et al., 1998). In addition, there is evidence that mTEC development and proliferation require CD4+or CD8+single positive (SP) thymocytes: mTEC do not develop in mice lacking mature T cells, (Shoreset al., 1994) whereas transplanted mature SP T cells induce a thymic medullary structure in SCID mice (Surhet al., 1992). These results suggest that the distribution of maturing thymocyte subpopulations may affect thymic architecture and that analysis of the intrathymic migration of hematopoietic and epithelial components is important in understanding thymic organogenesis. Thymocyte subpopulations express distinct chemokine receptor patterns, in part mandating their differential intrathymic migration patterns (Ansel and Cyster, 2001). For example, DN1 cells migrate to the thymic capsule in response to CXCR4 ligand (CXCL12/SDF1) produced by the ORY-1001(trans) thymic cortex (Plotkinet al., 2003). The CD44+CD25intCD4CD8(DN1-2 intermediate) cells respond to CCR7 ligands (CCL19/MIP3 and CCL21/6Ckine; CCL19/21) fostering migration to and retention in the subcapsular zone (Misslitzet al., 2004). Although resting CD4+CD8+double positive (DP) thymocytes are CCR9+, they do not respond to CCR9 ligand (CCL25; TECK); however, activated DP thymocytes expressing both CCR9 and CCR7 respond to CCL25 and CCL19/21. Mature CD4+CD8or CD4CD8+single positive (SP) thymocytes respond to CCL19/21 and migrate to the medulla where tissue-specific self-peptide/MHC complexes are provided to the maturing thymocytes, further facilitating removal of potentially autoreactive T cells (Uenoet al., 2004). It is clear that there are significant gaps in our current understanding of the signals mediated by chemokines and their receptors during thymic development. For example, CCL19/21 effects are pleiotropic, directing DN1-2 intermediate thymocytes to move to the subcapsullary zone, transitioning DP/SP thymocytes to move to the medulla, and mature SP thymocytes to exit the thymus (Uenoet al., 2002;Misslitzet al., 2004;Uenoet al., 2004). Furthermore, activated DP thymocytes respond to CCL25 expressed in the cortex and to CCL19/21 expressed in the medulla. Interestingly, CCL25 is provided by the cTEC (Wurbelet al., 2000) ORY-1001(trans) whereas CCL19/21 are expressed both in the medulla and the thymic capsule (Misslitzet al., 2004;Uenoet al., 2004). During positive selection, the maturing thymocytes are capable of receiving both signals because activated CD69+DP thymocytes express CCR7 and CCR9. Nevertheless, after positive selection, these thymocytes migrate rapidly to the medulla, despite the CCL25 cortical gradient (Uehara 2002). Therefore, a fundamental issue is to understand how activated thymocytes repress CCR9 signaling to allow their efficient CCR7-mediated migration to the medulla. As these complex chemokine networks suggest the existence of additional molecular pathways that regulate intrathymic migration of DP and SP thymocytes, we examined the global gene expression patterns of sorted developing thymocytes in order to identify such thymocyte subset-restricted molecules. We found thatPlxnd1mRNA is highly expressed in DP thymocytes and rapidly repressed thereafter. Recently, plexinD1 together with its ligand, semaphorin 3E (sema3E), was shown to play a critical role in directing angiogenesis (Krugeret al., 2005).Plxnd1deficiency results in defective developmental separation of the aorta and the pulmonary artery as well as inappropriate infiltration of blood vessels into H3FK somites during embryogenesis (Gitleret al., 2004;Guet al., 2005). Given the precisely-regulated expression ofPlxnd1in thymocytes, we hypothesized that the directional control exerted by other plexins and semaphorins during neuronal axon pathfinding (Krugeret al., 2005) and by PlexinD1 in angiogenesis.