Transgenic Expression of Numb Inhibits Notch Signaling in Immature Thymocytes But Does Not Alter T Cell Fate Specification

Abstract

The conserved adaptor protein Numb is an intrinsic cell fate determinant that functions by antagonizing Notch-mediated signal transduction. The Notch family of membrane receptors controls cell survival and cell fate determination in a variety of organ systems and species. Recent studies have identified a role for mammalian Notch-1 signals at multiple stages of T lymphocyte development. We have examined the role of mammalian Numb (mNumb) as a Notch regulator and cell fate determinant during T cell development. Transgenic overexpression of mNumb under the control of the Lck proximal promoter reduced expression of several Notch-1 target genes, indicating that mNumb antagonizes Notch-1 signaling in vivo. However, thymocyte development, cell cycle, and survival were unperturbed by mNumb overexpression, even though transgenic Numb was expressed at an early stage in thymocyte development (CD4−CD8−CD3− cells that were CD44+CD25+ or CD44−CD25+; double-negative 2/3). Moreover, bone marrow from mNumb transgenic mice showed no defects in thymopoiesis in competitive repopulation experiments. Our results suggest that mNumb functions as a Notch-1 antagonist in immature thymocytes, but that suppression of Notch-1 signaling at this stage does not alter γδ/αβ or CD4/CD8 T cell fate specification.

Normal Hemopoiesis and Lymphopoiesis in the Combined Absence of Numb and Numblike

“…1A). Although both Nb and Nbl are expressed in thymocytes as expected from previously published results (18,24,25) (Fig. 1A), only Nb is detectable in total BM (Fig.…”

“…As the Nb homolog Nbl is able to substitute for Nb in neuronal precursors (3), it is conceivable that they may also play redundant roles in other developing organs such as the hemopoietic system. Consistent with this hypothesis, Nb and Nbl have been shown to be expressed (by RT-PCR or Northern or Western blots) in hemopoietic tissues such as the thymus, spleen, lymph nodes, and PBLs in both mouse and human models (18,(22)(23)(24)(25). To determine whether Nb and Nbl are also expressed in the most immature hemopoietic cells, we performed semiquantitative RT-PCR on RNA isolated from BM from which all hemopoietic lineage cells originate (Fig.…”

“…Conditional deletion of Nb alone in the BM showed no phenotype in any hemopoietic lineage (data not shown), possibly due to the previously documented redundancy of Nbl in tissues where both genes are expressed, such as in the developing brain (21,24) and lymphoid tissues (18,24,25) (Fig. 1A) and in BM (Fig.…”

Analysis of Notch1 Function by In Vitro T Cell Differentiation ofPax5Mutant Lymphoid Progenitors

“…1). Pax5 Ϫ/Ϫ pro-B cells furthermore do not express transcripts for Numb and Nrarp, which are negative modifiers of intracellular Notch signaling (16,25,45). However, Pax5…”

Asymmetric Division in the Immuno-Hematopoietic System

“…However, the first studies related to asymmetric division in the thymus used either transgenic or knockout mice to over-express or delete Numb (French et al 2002;Anderson et al 2005;Wilson et al 2007). In these studies, investigators used classical assessments of thymocyte differentiation in order to determine whether or not Numb played a role in thymocyte differentiation (they never examined asymmetric division).…”