The Notch signaling pathway is involved throughout the hematolymphoid system, from the generation of definitive HSCs to the differentiation of peripheral T and B cells (57
). In this study, our data provide in vivo evidence that an E3 ubiquitin ligase of Notch signaling, Mib1, is required for lymphopoiesis, including T lineage commitment and MZB cell development. Conditional inactivation of the Mib1
gene caused a block of T lineage commitment and MZB cell development, whereas their development was not perturbed in the other three mutant mice with inactivation of Mib2
, and Neur2
, and even in the Neur1/2
double KO mice. More importantly, reciprocal BMT experiments revealed that Mib1 in the thymic and splenic microenvironments is essential for Notch signaling to the hematopoietic progenitors. Although the Notch ligands and the E3 ligases are expressed in both hematopoietic progenitors and their microenvironments () (59
), the defective Notch activation of hematopoietic progenitors and the failure of Notch ligand endocytosis were observed in the Mib1-null microenvironment. These results demonstrate that Notch signaling in the lymphopoietic niches to progenitors, but not the interactions between hematopoietic cells, is required for lymphopoiesis. In addition, the block in T cell development and the defect in the endocytosis of Dll1 were also observed in OP9-DL1 cell by Mib1 knockdown. Collectively, our study provides the first in vivo evidence that Mib1, in the lymphopoietic niches, controls T lineage commitment and MZB cell development by regulating Notch signaling.
The Notch signaling pathway is conserved in all metazoans. So far, four E3 ubiquitin-ligases, Mib1, Mib2, Neur1, and Neur2, which regulate the Notch ligands, have been identified in vertebrates, and three, dMib1, dMib2, and dNeur, have been found in D. melanogaster
). In D. melanogaster
, mutations in dNeur
result in a variety of developmental defects that closely resemble those of Notch mutants and other Notch pathway mutants (60
). However, mice with a disrupted Neur1
gene exhibit no abnormal cell fate specifications during neurogenesis and somitogenesis, two processes involving Notch signaling (35
). This discrepancy might be caused by the functional redundancy provided by Neur2. We originally expected a subset of Notch phenotypes in the Neur1/2
double KO mice because most mutant mice with disruptions in the key components of the Notch signaling pathway, such as Dll1, Dll4, Jag2, Notch1, Notch2, lunatic fringe, RBP-Jκ, MAML1, and MINT, exhibit a subset of defects in lymphopoiesis (58
). In this study, however, T and B cell development was not disturbed in the Neur1/2
double KO mice, demonstrating that Neur1/2
is not involved in these cell fate decisions.
In addition to Neur, Mib1 has been identified as another E3 ubiquitin ligase that interacts with Delta to promote its ubiquitination and internalization in the signal-sending cells, using zebrafish mutant models (21
). In D. melanogaster
, dMib1 is involved in regulating Notch ligand endocytosis, and the ectopic expression of dNeur bypasses the requirement for dMib1, suggesting that they appear to be interchangeable in mediating the ubiquitination and internalization of the Notch ligand (23
). However, dMib1 and dNeur are expressed in different patterns (23
), suggesting that both dNeur and dMib1 regulate Notch ligand endocytosis in different contexts of Notch-dependent cell fate decisions. In contrast to the results in D. melanogaster
-null mouse embryos showed completely defective Notch activation in terms of NICD generation and Notch-target gene expression (26
), whereas the Neur1/2
double KO mice did not show any gross defects in mammalian development (34
). Consistent with these findings, a developmental block in T and MZB cell development, which is a prototype of a Notch signaling defect, was observed only in Mib1
-null mice, suggesting an obligatory role of Mib1 in mammalian T and MZB cell development.
In addition to Mib1, we previously identified Mib2 as an E3 ligase that regulates Delta (27
). Overexpression of Mib2 rescues both the neuronal and vascular defects in the zebrafish Mibta52b
), suggesting that they are interchangeable in mediating the ubiquitination and internalization of the Notch ligand. Mib2 is highly expressed in adult tissues, whereas Mib1 is expressed in both embryonic and adult tissues (27
). Therefore, although Mib1 is an essential core component of the mammalian Notch pathway that controls multiple Notch ligands (26
), it is necessary to determine whether Mib1 regulates multiple ligands beyond embryonic development. The existence of Mib2, which has functional similarities to Mib1 and is highly expressed in adult tissues with Mib1, brings us to speculate that both Mib1 and Mib2 might have functional redundancy in the adult tissues (27
). However, in vivo studies using KO mice have revealed the essential role of Mib1 in Notch-mediated lymphocyte development beyond embryonic development.
We previously found that Mib1 is an essential regulator for generating functional Notch ligands to activate Notch signaling (26
). In this study, the conditional inactivation of Mib1
disturbed both T lineage commitment and MZB cell specification, suggesting that Dll1 and Dll4 should be nonfunctional. Indeed, the BM transplantation experiments using the TNR mice (51
) demonstrate that the Mib1-null thymic microenvironment cannot initiate Notch signaling to thymic progenitors. Moreover, our reciprocal BM transplantation experiments revealed that the Mib1-null lymphopoietic niches cannot support T lineage commitment and MZB cell specification. This inability of Mib1-null microenvironments might be caused by the malfunction of Dll1 and Dll4. Consistent with this finding, the Dll1 was accumulated in the MMTV-Cre;Mib1f/f
thymus, although it is unlikely that most of Dll1 was present at the plasma membrane. This abnormal localization of Dll1 might result from the continual accumulation of Dll1 in the ER, Golgi, or other organelle in the absence of Mib1. Furthermore, the block in T cell development and the failure of Dll1 endocytosis were also found in OP9-DL1 cells by Mib1 knockdown, which recapitulates the defect in T cell development observed in the MMTV-Cre;Mib1f/f
mice. Collectively, our data suggest that Mib1 controls Notch signaling in the thymic progenitors from the thymic stromal cells, through the regulation of Dll1 and Dll4.
We previously proposed that Mib1 and Neur2 may play a cooperative role in the endocytic pathway of Delta using COS-7 cell lines (25
). In this study, however, the endocytosis of Dll1 was impaired in the thymi from only Mib1
-null mice but not Neur1−/−
, or Neur1−/−
mice. In addition, Mib2−/−
mice also showed normal endocytosis of Dll1, despite Mib2 readily inducing its endocytosis in vitro (27
). This discrepancy might be caused by the difference between in vivo and in vitro systems. Our in vivo study using KO mice would represent more relevant physiology of the Notch ligand endocytosis than in vitro observation.
Many studies have reported that maintenance of HSCs and regulation of their self-renewal and differentiation depends on their specific microenvironment (66
). Notch ligand–receptor interactions between the BM microenvironment and hematopoietic cells are thought to have a role in the maintenance of HSCs (68
). In this paper, we have clearly shown that Notch signaling between the lymphopoietic niches and hematopoietic cells is required for T and MZB cell specification. This study will help elucidate the exact cellular sources in the lymphopoietic niches that trigger Notch signaling to their progenitors.