The endocytic adaptor Numb regulates thymus size by modulating pre-TCR signaling during asymmetric division

Abstract

Stem cells must proliferate and differentiate to generate the lineages that shape mature organs; understanding these 2 processes and their interaction is one of the central themes in current biomedicine. An intriguing aspect is asymmetric division, by which 2 daughter cells with different fates are generated. Several cell fate determinants participate in asymmetric division, with the endocytic adaptor Numb as the best-known example. Here, we have explored the role of asymmetric division in thymocyte development, visualizing the differential segregation of Numb and pre-TCR in thymic precursors. Analysis of mice where Numb had been inhibited by expressing a dominant negative revealed enhanced pre-T-cell receptor (TCR) signaling and a smaller thymus. Conversely, Numb overexpression resulted in loss of asymmetric division and a larger thymus. The conclusion is that Numb determines the levels of pre-TCR signaling in dividing thymocytes and, ultimately, the size of the pool from which mature T lymphocytes are selected. IntroductionThe mammalian thymus contains different cell types originated from initially equivalent precursors that undergo several rounds of division. To ensure correct numbers of mature lineage-specific cells in adult organs, precursors divide asymmetrically, generating 2 sister cells with different fates. 1,2 One of the "cell fate determinants" asymmetrically segregated is Numb, a plasma membrane-associated protein that contains a phosphotyrosine binding (PTB) domain and antagonizes Notch signaling. Numb function was first described in Drosophila sensory organ precursors, 3 where loss of Numb resulted in both daughter cells adopting the fate of the cell that normally inherits Notch, whereas the opposite was caused by Numb overexpression. [4][5][6] In mammalians, asymmetric division has been studied most extensively during neurogenesis. 7 Deletion of Numb and its homologue Numblike resulted in loss of neural progenitors and block of neurogenesis, 8 as a result of progenitor overdifferentiation and death of young neurons. In other systems, Numb binds to integrins 9 and Src family kinases, 10 mediates receptor internalization, 11,12 and participates in clathrin-dependent endosomal association. 13 Thus, Numb plays multiple and important roles in development and signaling; however, little is known about its role in the thymus.T lymphocytes develop from precursors that undergo a series of cell fate decisions, resulting in differentiation into single positive (SP) thymocytes, the immediate precursors of fully functional CD4 and CD8 T lymphocytes. 14,15 The initial CD4 Ϫ CD8 Ϫ double negative (DN) stages are influenced by signals from the pre-T-cell receptor (TCR) [16][17][18] and Notch. 19,20 Only DN thymocytes receiving proper pre-TCR and Notch signals are able to evolve into the CD4 ϩ CD8 ϩ double positive (DP) ␣␤ lineage stage. Pre-TCR internalization and degradation is very important for correct signaling. [21][22][23] As a consequence of pre-TCR signaling, DN thymocytes proliferate, enabling normal nu...

Notch Receptor-Ligand Interactions During T Cell Development, a Ligand Endocytosis-Driven Mechanism

“…This is in contrast to other studies, in which overexpression of Numb specifically in T cells causes a reduction in Notch activity (French et al 2002); however, recent studies using overexpression of full length Numb resulted in a larger thymus and loss of pre-TCR signaling, whereas overexpression of a dominant-negative Numb resulted in a smaller thymus and enhanced pre-TCR signaling and differentiation (Aguado et al 2010) indicating that the role of Numb in T cell development still requires further analysis. ADAM proteases are required for both Notch receptor and ligand proteolytic processing.…”

Polarized Cells, Polarized Views: Asymmetric Cell Division in Hematopoietic Cells

“…Interestingly, depletion of Scribble affects DN3 thymocyte clustering by limiting the polarization of the integrin ICAM-1/LFA-1 (77, 78). In a study by Aguado and colleagues, the transgenic expression of wildtype or dominant negative forms of Numb result in altered DN3 thymocyte pre-TCR signaling, proliferation, and differentiation (79). Asymmetry of Numb was also proposed by this group as a mechanism for these signaling and fate differences, but asymmetry was not rigorously assessed.…”

New insights into the T cell synapse from single molecule techniques

“…However, many conclusions about the localization of signalling intermediates have been based on imaging methods that might not resolve SSVs from plasma membranes. Signalling by receptors in endosomes, such as the pre-TCR or some Toll-like receptors, is a well-described process 94 . The participation of SSVs in T cell signalling based on a docking mechanism is a novel situation and creates many new possibilities for the compartmentalization of signalling that need to be explored.…”