Katsuto Hozumi

Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear.

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27Kip1 (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.

Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo

Notch receptors and their ligands contribute to many developmental systems, but it is not apparent how they function after birth, as their null mutants develop severe defects during embryogenesis. Here we used the Cre-loxP system to delete the Delta-like 1 gene (Dll1) after birth and demonstrated the complete disappearance of splenic marginal zone B cells in Dll1-null mice. In contrast, T cell development was unaffected. These results demonstrated that Dll1 was dispensable as a ligand for Notch1 at the branch point of T cell–B cell development but was essential for the generation of marginal zone B cells. Thus, Notch signaling is essential for lymphocyte development in vivo, but there is a redundancy of Notch-Notch ligand signaling that can drive T cell development within the thymus.

Jagged 1 is a β-catenin target gene required for ectopic hair follicle formation in adult epidermis

The Wnt and Notch signalling pathways regulate hair follicle maintenance, but how they intersect is unknown. We show that Notch signalling is active in the hair follicle pre-cortex, a region of high Wnt activity, where commitment to hair lineages occurs. Deletion of jagged 1 (Jag1) results in inhibition of the hair growth cycle and conversion of hair follicles into cysts of cells undergoing interfollicular epidermal differentiation. Conversely, activation of Notch in adult epidermis triggers expansion of the base of the hair follicle, sebaceous gland enlargement and abnormal clumping of the follicles. In adult epidermis, the induction of new hair follicle formation by β-catenin is prevented by blocking Notch signalling pharmacologically or through Jag1 deletion. Conversely, activation of both pathways accelerates growth and differentiation of ectopic follicles.β -catenin stimulates Notch signalling by inducing Jag1 transcription. We conclude that the Notch pathway acts downstream of the Wnt/β-catenin pathway to determine epidermal cell fate.

The Runx1 Transcription Factor Inhibits the Differentiation of Naive CD4+ T Cells into the Th2 Lineage by Repressing GATA3 Expression

Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

Notch controls the survival of memory CD4+ T cells by regulating glucose uptake

CD4+ T cells differentiate into memory T cells that protect the host from subsequent infection. In contrast, autoreactive memory CD4+ T cells harm the body by persisting in the tissues. The underlying pathways controlling the maintenance of memory CD4+ T cells remain undefined. We show here that memory CD4+ T cell survival is impaired in the absence of the Notch signaling protein known as recombination signal binding protein for immunoglobulin κ J region (Rbpj). Treatment of mice with a Notch inhibitor reduced memory CD4+ T cell numbers and prevented the recurrent induction of experimental autoimmune encephalomyelitis. Rbpj-deficient CD4+ memory T cells exhibit reduced glucose uptake due to impaired AKT phosphorylation, resulting in low Glut1 expression. Treating mice with pyruvic acid, which bypasses glucose uptake and supplies the metabolite downstream of glucose uptake, inhibited the decrease of autoimmune memory CD4+ T cells in the absence of Notch signaling, suggesting memory CD4+ T cell survival relies on glucose metabolism. Together, these data define a central role for Notch signaling in maintaining memory CD4+ T cells through the regulation of glucose uptake.

Mib-Jag1-Notch signalling regulates patterning and structural roles of the notochord by controlling cell-fate decisions

In the developing embryo, cell-cell signalling is necessary for tissue patterning and structural organization. During midline development, the notochord plays roles in the patterning of its surrounding tissues while forming the axial structure; however, how these patterning and structural roles are coordinated remains elusive. Here, we identify a mechanism by which Notch signalling regulates the patterning activities and structural integrity of the notochord. We found that Mind bomb (Mib) ubiquitylates Jagged 1 (Jag1) and is essential in the signal-emitting cells for Jag1 to activate Notch signalling. In zebrafish, loss- and gain-of-function analyses showed that Mib-Jag1-Notch signalling favours the development of non-vacuolated cells at the expense of vacuolated cells in the notochord. This leads to changes in the peri-notochordal basement membrane formation and patterning surrounding the muscle pioneer cells. These data reveal a previously unrecognized mechanism regulating the patterning and structural roles of the notochord by Mib-Jag1-Notch signalling-mediated cell-fate determination.

Selection of differentiating cells by different levels of delta-like 1 among neural precursor cells in the developing mouse telencephalon

During the neurogenic phase of mammalian brain development, only a subpopulation of neural precursor cells (NPCs) differentiates into neurons. The mechanisms underlying this selection remain unclear. Here we provide evidence that the Notch-Delta pathway plays an important role in this selection in the developing mouse telencephalon. We found that the expression patterns of the Notch ligand delta-like 1 (Dll1) and of the active form of Notch1 were mutually exclusive and segregated into distinct NPC subpopulations in the ventricular zone of the telencephalon. When Dll1 was overexpressed in a small, but not a large, proportion of NPCs, these cells underwent neuronal differentiation in vitro and in vivo. This Dll1-induced neuronal differentiation did not occur when cells were plated at lower densities in an in vitro culture. Importantly, conditional deletion of the Dll1 gene in a small proportion of NPCs reduced neurogenesis in vivo, whereas deletion in a large proportion promoted premature neurogenesis. These results support the notion that different levels of Dll1 expression determine the fate of NPCs through cell-cell interactions, most likely through the Notch-Delta lateral inhibitory signaling pathway, thus contributing to the selection of differentiating cells.

Pre-TCR signaling regulates IL-7 receptor alpha expression promoting thymocyte survival at the transition from the double-negative to double-positive stage.

The pre‐T cell receptor (pre‐TCR) and IL‐7 receptor (IL‐7R) are critical mediators of survival, proliferation and differentiation in immature thymocytes. Here we show that pre‐TCR signaling directly maintains IL‐7Rα expression as developing thymocytes undergo β‐selection. Inhibition of IL‐7/IL‐7R signaling in (CD44–CD25–) DN4 cells results in decreased generation of double‐positive thymocytes due to increased death of rapidly proliferating β‐selected cells. Thus, we identify a mechanism by which pre‐TCR signaling controls the selective survival of TCRβ+ thymocytes, and define a further stage of T cell differentiation in which signaling from a TCR regulates the ability of that cell to respond to cytokine.

Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis

Stem cells often divide asymmetrically to produce one stem cell and one differentiating cell, thus maintaining the stem cell pool. Although neural stem cells (NSCs) in the adult mouse subventricular zone have been suggested to divide asymmetrically, intrinsic cell fate determinants for asymmetric NSC division are largely unknown. Stem cell niches are important for stem cell maintenance, but the niche for the maintenance of adult quiescent NSCs has remained obscure. Here we show that the Notch ligand Delta-like 1 (Dll1) is required to maintain quiescent NSCs in the adult mouse subventricular zone. Dll1 protein is induced in activated NSCs and segregates to one daughter cell during mitosis. Dll1-expressing cells reside in close proximity to quiescent NSCs, suggesting a feedback signal for NSC maintenance by their sister cells and progeny. Our data suggest a model in which NSCs produce their own niche cells for their maintenance through asymmetric Dll1 inheritance at mitosis.

Active Form of Notch Members Can Enforce T Lymphopoiesis on Lymphoid Progenitors in the Monolayer Culture Specific for B Cell Development

The in vitro induction of T lymphopoiesis needs the precise stereoscopic structure of thymus tissues as seen in fetal thymus organ culture. In this study, we demonstrated for the first time that the introduction of the intracellular region of Notch1 can induce T cells expressing TCR without any thymic environment. In the coculture on the monolayer of OP-9, which was originally known to support B cell specific development, hemopoietic progenitors developed into Thy-1+CD25+ T lineage cells if the progenitor cells were infected with the retrovirus containing Notch1 intracellular domains. The Thy-1+ cells progressed to a further developmental stage, CD4 and CD8 double-positive cells expressing TCR on the cell surface, if they were further cultured on OP-9 or in the thymus. However, T cell induction by intracellular Notch1 failed unless both OP-9 and IL-7 were present. It is notable that Notch2 and Notch3 showed an effect on T lymphopoiesis similar to that of Notch1. These results indicate that in vitro T lymphopoiesis is inducible by signaling via Notch family members in a lineage-specific manner but shares other stroma-derived factors including IL-7 with B lymphopoiesis.