Genes and Immunity (2006) 7, 101–112 & 2006 Nature Publishing Group All rights reserved 1466-4879/06 $30.00 www.nature.com/gene ORIGINAL ARTICLE Identification of novel genes and transcription factors involved in spleen, thymus and immunological development and function BG Hoffman1, KL Williams2, AH Tien1,VLu1, T Ruiz de Algara1, JP-Y Ting2 and CD Helgason1,3 1BC Cancer Agency, Vancouver, BC, Canada; 2University of North Carolina Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA and 3Department of Surgery, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada We constructed and analyzed six serial analysis of gene expression (SAGE) libraries to identify genes with previously uncharacterized roles in spleen or thymus development. A total of 625 070 tags were sequenced from the three spleen (embryonic day (E)15.5, E16.5 and adult) and three thymus (E15.5, E18.5 and adult) libraries. These tags corresponded to 83 182 tag types, which mapped unambiguously to 36 133 different genes. Genes over-represented in these libraries, compared to 115 mouse SAGE libraries (www.mouseatlas.org), included genes of known and unknown immunological or developmental relevance. The expression profiles of 11 genes with unknown roles in spleen and thymus development were validated using reverse transcription-qPCR. We further characterized the expression of one of these candidates, RIKEN cDNA 9230105E10 that encodes a murine homolog of Trim5a, in numerous adult tissues and immune cell types. In addition, we demonstrate that transcript levels are upregulated in response to TLR stimulation of plasmacytoid dendritic cells and macrophages. This work provides the first evidence of regulated and cell type-specific expression of this gene. In addition, these observations suggest that the SAGE libraries provide an important resource for further investigations into the molecular mechanisms regulating spleen and thymus organogenesis, as well as the development of immunological competence. Genes and Immunity (2006) 7, 101–112. doi:10.1038/sj.gene.6364270; published online 15 December 2005 Keywords: SAGE; Trim5a; transcription factor Introduction developing spleen can no longer be seen at this time in Hox11À/À, capsulinÀ/À and Bapx1À/À mice,1–3,5 confirm- Comparatively little is known about the molecular ing the critical role of these transcription factors in spleen mechanisms regulating the development of the spleen organogenesis. The first hematopoietic cells begin to and thymus. Considerably more attention has been appear in the spleen anlage, and between E13.5 and focused on elucidating the mechanisms regulating approximately E16.5, the spleen is a site of hematopoiesis lymphoid cell development and function, although it is in the fetus. Shortly after E16.5 hematopoiesis migrates to likely that many genes critical to these processes remain the bone marrow and lymphoid precursors begin to to be discovered. infiltrate the spleen. Thereafter, the final organization of Development of the mouse spleen begins just before the spleen is established with the formation of the white embryonic day 11.5 (E11.5) with condensation of the pulp, containing B-cell follicles, T cells as well as mesenchyme underlying the dorsal mesogastrium lymphocytes and antigen-presenting dendritic cells epithelium.1 The genetic mechanisms underlying the (DCs) in the marginal zone, and the red pulp, which is generation of these initial splenic precursors are essen- composed of a complex system of blood vessels that tially unknown. By E11.5, the expression of several allow the removal of old or damaged red blood cells.6,7 transcription factors essential to spleen organogenesis Thymus development begins as early as E10.5 with the including Hox11, Nkx2.5, capsulin/Pod-1, Wt1 and Bapx1 proliferation of cells in the posterior part of the third can be detected.1–3 The molecular hierarchy regulating pharyngeal pouch.8,9 Subsequently, thymic precursor expression of these transcription factors, and the path- cells migrate and fuse at the midline. Interactions ways they function within, are only beginning to be between the thymic epithelium and the surrounding established.1,4 By E13.5, the splenic primordium is visible neural crest-derived mesenchyme play a key role in as a ridge of cells on the dorsal stomach. A normally expansion of thymic precursors.9,10 Several studies have highlighted the importance of transcription factors such as Foxn1, Hoxa3, Pax1 and Pax9 in early thymus Correspondence: Dr CD Helgason, British Columbia Cancer development.8,9,11–17 These studies point to a hierarchy Agency, 675 West 10th Avenue, Vancouver, B.C., V5Z 1L3, Canada. E-mail: [email protected] of transcription factor expression, with Foxn1 and Hoxa3 Received 19 September 2005; revised 12 October 2005; accepted 13 playing a role in early thymic development and Pax9 and October 2005; published online 15 December 2005 Pax1 becoming involved subsequently.8,9 At roughly the SAGE analysis of spleen and thymus development BG Hoffman et al 102 same time that Foxn1 expression becomes critical to candidate uncharacterized genes, a mouse homolog of thymic development (E11.5), hematopoietic precursors the primate Trim5a HIV-1 restriction factor, was further begin to invade the thymic primordium. Interestingly, assessed in numerous adult tissues as well as various Foxn1À/À mice lack expression of the chemokines TECK immune cell types including macrophages, DCs and and SDF-1, suggesting that these molecules are targets of various T-cell populations. Our results demonstrate Foxn1 and that they are critical in attracting prothymo- regulated expression of this transcript in macrophages cytes to the thymus.8 At E11.5, the thymic epithelium is and Flt-3L-derived DC populations in response to TLR not fully differentiated and is unable to support T-cell stimulation. The libraries created and described herein development. By E14.5, concomitant with maturation of provide a unique and valuable resource for the identi- the thymic epithelium, T-cell precursors become fully fication of developmentally and immunologically rele- committed to the T-cell lineage at least in part via the vant transcripts in the spleen and thymus. Moreover, we Notch signaling pathway.18–23 Thymocyte interactions provide the first evidence that expression of an im- with the thymic epithelium and mesenchyme are critical munologically relevant transcript identified in these to the proper development of both the thymus and the libraries is differentially regulated. thymocytes. Lineage-committed T cells drive the estab- lishment of the appropriate architecture of cortical thymic epithelial cells. Subsequently, the final maturation of T cells induces the expansion and organization of Results medullary thymic epithelial cells. Overview of the libraries Serial analysis of gene expression (SAGE) analysis, like A total of 625 070 tags were sequenced to generate the six microarrays, provides a quantitative analysis of gene LongSAGE spleen and thymus libraries (available at expression profiles. However, it has the additional www.mouseatlas.org or http://cgap.nci.nih.gov/SAGE). advantage that it permits the identification of novel These include three spleen libraries from: E15.5 when the transcripts, which has been improved by the develop- spleen is predominantly erythropoietic, E16.5 when ment of a LongSAGE protocol, which generates 21 mers lymphocytes begin to appear in this tissue and adult that enhances the efficiency and confidence of tag-to- (84 days post-natal (dpn)) when the spleen is fully gene mapping. SAGE also has the added benefit that the mature and functional. Similarly, we generated three data are digital and thus easily shared among investiga- thymus libraries from: E15.5 when a mature thymic tors and compared across different experiments and epithelium is present and T-cell precursors become fully tissues.24,25 committed, E18.5 when mature T cells are first estab- The analysis of expression patterns associated with lished and from 84 dpn when the thymus is fully mature different stages of spleen and thymus development is and functional. A summary of the libraries is shown in expected to further facilitate our understanding of the Table 1. genetic cascade required for their development. In Analysis of these libraries revealed expression of addition, such studies are likely to provide insights into 83 182 different tag types after stringent quality selection: the numerous transcription factors, cell surface proteins 47 727 in the spleen libraries (58% unique to the spleen and signaling pathway components involved in the libraries as compared to the thymus libraries) and 55 282 maturation and function of lymphocytes. We thus in the thymus libraries (64% unique). Of these tags, 45% created six SAGE libraries representing critical stages of (37 202) mapped to known transcripts using the Refseq, spleen (E15.5 and E16.5) and thymus (E15.5 and E18.5) MGC and Ensembl transcript databases, 23% (19 214) to development, as well as libraries from the corresponding known genes using the Ensembl genes database, 12% adult tissues. Here, we describe the analyses of these (9626) to the genome using the Golden path database and libraries focusing on the identification of transcription 0.1% (100) to the mitochondrial genome or to noncoding factors, signaling pathway members and uncharacter- genes using the Genebank database. The remaining 20% ized genes. We next validated the expression of seven (17 040) of the tags could not be