Transcription factor Tlx1 marks a subset of lymphoid tissue organizer-like mesenchymal progenitor cells in the neonatal spleen.

The spleen is comprised of spatially distinct compartments whose functions, such as immune responses and removal of aged red blood cells, are tightly controlled by the non-hematopoietic stromal cells that provide regionally-restricted signals to properly activate hematopoietic cells residing in each area. However, information regarding the ontogeny and relationships of the different stromal cell types remains limited. Here we have used in vivo lineage tracing analysis and in vitro mesenchymal stromal cell assays and found that Tlx1, a transcription factor essential for embryonic spleen organogenesis, marks neonatal stromal cells that are selectively localized in the spleen and retain mesenchymal progenitor potential to differentiate into mature follicular dendritic cells, fibroblastic reticular cells and marginal reticular cells. Furthermore, by establishing a novel three-dimensional cell culture system that enables maintenance of Tlx1-expressing cells in vitro, we discovered that signals from the lymphotoxin ß receptor and TNF receptor promote differentiation of these cells to express MAdCAM-1, CCL19 and CXCL13, representative functional molecules expressed by different subsets of mature stromal cells in the spleen. Taken together, these findings indicate that mesenchymal progenitor cells expressing Tlx1 are a subset of lymphoid tissue organizer-like cells selectively found in the neonatal spleen.

Niche-induced extramedullary hematopoiesis in the spleen is regulated by the transcription factor Tlx1.

Extramedullary hematopoiesis (EMH) in postnatal life is a pathological process in which the differentiation of hematopoietic stem/progenitor cells (HSPCs) occurs outside the bone marrow (BM) to respond to hematopoietic emergencies. The spleen is a major site for EMH; however, the cellular and molecular nature of the stromal cell components supporting HSPC maintenance, the niche for EMH in the spleen remain poorly understood compared to the growing understanding of the BM niche at the steady-state as well as in emergency hematopoiesis. In the present study, we demonstrate that mesenchymal progenitor-like cells expressing Tlx1, an essential transcription factor for spleen organogenesis, and selectively localized in the perifollicular region of the red pulp of the spleen, are a major source of HSPC niche factors. Consistently, overexpression of Tlx1 in situ induces EMH, which is associated with mobilization of HSPC into the circulation and their recruitment into the spleen where they proliferate and differentiate. The alterations in the splenic microenvironment induced by Tlx1 overexpression in situ phenocopy lipopolysaccharide (LPS)-induced EMH, and the conditional loss of Tlx1 abolished LPS-induced splenic EMH. These findings indicate that activation of Tlx1 expression in the postnatal splenic mesenchymal cells is critical for the development of splenic EMH.

Fluorimetric assay for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate.

An easy fluorimetric assay for measuring D-amino acid oxidase (DAAO) activity by using one of the D-amino acids--D-kynurenine (D-KYN)--as a substrate was applied to assess DAAO activity in the cerebrum, cerebellum, and brainstem of Sprague-Dawley (SD) male rats. In this assay, DAAO produces kynurenic acid (KYNA) from D-KYN, and the fluorescence originating from KYNA can then be used to evaluate DAAO activity. Here, pellet fractions obtained by centrifugation of brain homogenates were allowed to react enzymatically with D-KYN. The addition of specific DAAO inhibitors, such as 3-methylpyrazole-5-carboxylic acid and 4H-thieno [3, 2-b] pyrrole-5-carboxylic acid (Compound 8), significantly attenuated the fluorescence intensity of KYNA, suggesting that DAAO present in the rat brain homogenates was responsible for the production of KYNA. In contrast, an inhibitor of aminotransferase (AT), aminooxyacetic acid, did not decrease KYNA production from D-KYN, meaning that AT could not metabolize D-KYN to KYNA under the present conditions. Moreover, the DAAO activity measured by the proposed assay correlated well with DAAO mRNA expression (r = 0.9982) determined by real-time polymerase chain reaction. Taken together, these findings show that the proposed fluorimetric assay can be used to evaluate DAAO activity in rat brain.