A Simplified Approach to Evaluating T Cell Development by Flow Cytometry.

T cell development is a complex multistep process that requires the coordinated activation of distinct signaling responses and the regulated progression of developing cells (thymocytes) through key stages of maturation. Although sophisticated techniques such as fetal thymus organ culture, in vitro thymocyte culture, and multiparameter flow cytometry-based cell sorting are now widely employed to evaluate thymocyte maturation by experienced laboratories, defects in T cell development can usually be identified with relatively simple flow cytometry screening methods. Here, we provide a basic protocol for assessment of T cell development that will enable laboratories with access to a multi-laser flow cytometer to screen mouse strains, including those generated from embryonic stem cells with targeted gene mutations, for thymocyte maturation defects.

Ldb1 is required for Lmo2 oncogene-induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia.

Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12.

Proliferation is tightly regulated during T cell development, and is limited to immature CD4-CD8- thymocytes. The major proliferative event is initiated at the 'ß-selection' stage following successful rearrangement of Tcrß, and is triggered by and dependent on concurrent signaling by Notch and the pre-T cell receptor (TCR); however, it is unclear how these signals cooperate to promote cell proliferation. Here, we found that ß-selection-associated proliferation required the combined activity of two Skp-cullin-F-box (SCF) ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for polyubiquitination and proteasomal degradation. We found that Notch signals induced the transcription of Fbxl1, whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of ß-selected thymocytes.

Assessment of T Cell Development by Flow Cytometry.

T cell development is a complex multistep process that requires the coordinated activation of distinct signaling responses and the regulated progression of developing cells (thymocytes) through key stages of maturation. Although sophisticated techniques such as fetal thymus organ culture, in vitro thymocyte culture, and multi-parameter flow cytometric analysis are now widely employed to evaluate thymocyte maturation by experienced laboratories, defects in T cell development can usually be identified with more simplified screening methods. Here, we provide a basic protocol for assessment of T cell development that will enable laboratories with access to a four parameter flow cytometer to screen mouse strains, including those generated from embryonic stem cells with targeted gene mutations, for thymocyte maturation defects.

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

A requirement for Lim domain binding protein 1 in erythropoiesis.

During erythrocyte development, the nuclear cofactor Lim domain binding protein 1 (Ldb1) functions as a core subunit of multiprotein DNA binding complexes that include the transcription factors Scl and Gata-1 and the Lim-only adapter Lmo2. Scl, Gata-1, and Lmo2 are each required for erythropoiesis, suggesting that Ldb1-nucleated transcription complexes regulate key steps during erythropoiesis. We documented a requirement for Ldb1 in erythropoiesis in mice. Analysis of ldb1(-/-) embryos revealed a critical requirement for Ldb1 during primitive erythropoiesis, and conditional inactivation of ldb1 at later stages of gestation and in adult mice demonstrated that Ldb1 is continuously required for both definitive erythropoiesis and megakaryopoiesis. Down-regulation of Ldb1 in erythroblasts inhibited the expression of multiple erythroid-specific and prosurvival genes. These results represent the first unequivocal demonstration of a role for Ldb1 in erythropoiesis in vivo and establish a critical function for Ldb1-nucleated complexes in regulating the erythroid/megakaryocyte transcriptional program.