Bcl11b is known to plays crucial roles in the development of several organs, including T cells, CNS, skin, and tooth. Bcl11b-deficient mice exhibit various developmental defects in these organs as follows.
4-1. T cells.
As described above, the defect in T-cell development given by Bcl11b deficiency is the phenotype firstly discovered. At present, defects have been identified at several distinct stages of development of thymocytes and T cells.3,22,43–47)
Figure A illustrates T-cell development in thymus (see below for details) and indicates the stages of developmental arrest given by loss of Bcl11b. Bcl11b is a unique transcription factor that specifically functions for T-cell identity maintenance and another transcription factor of this type is Tcf1 (T-cell factor 1).22,46–48)
The T cell specification pathway involves many different signalings, one of which is the signaling by the Notch1 receptors. The receptors are expressed by early progenitors, and activated to function upon interaction with cognate ligands (Delta-like proteins) expressed by thymic epithelial cells.49,50)
The review by Liu et al.1)
describe tissue-specific signals that direct developmental fates of thymocyte progenitors in the thymus. To avoid redundancy, I touch briefly on thymocyte development and roles for Bcl11b in controlling thymocyte differentiation and expansion.
T cells arise from hematopoietic progenitor cells that migrate from the bone marrow to the thymus, where they proliferate as thymocytes (Fig. A). Development of αβ T cells in the thymus proceeds through three major stages defined according to their expression pattern of CD4 and CD8 molecules on cell surface, i.e. in order of maturity, CD4−CD8− double negative (DN), CD4+CD8+ double positive (DP), and CD4+CD8− or CD4−CD8+ single positive cells (CD4SP or CD8SP cells, respectively). The CD4 and CD8 molecules are coreceptors of the T-cell receptor (TCR). Before DN thymocytes progress to the DP stage, they express CD8 but lack αβ TCR on cell surface. Those cells are highly proliferative and called immature CD8+ single positive (ISP) cells. Thymocytes at the DP stage express the αβ T-cell receptor (αβ TCR) complex after rearrangement at the TCRα locus, which allows engagement by intrathymic peptide/major histocompatibility complex (MHC) ligands. CD4 and CD8 molecules interact, respectively, with class II and I MHC molecules, thereby stabilizing or enhancing the interaction of TCRs with their MHC ligands. Co-expression of CD4 and CD8 at the DP stage allows thymocytes to receive optimal signals through either class I or II MHC-specific TCRs. Negative selection leads to death by apoptosis, while positive selection leads to thymocyte activation as evidenced by the upmodulation of activation markers, such as CD5 and CD69, and differentiation into SP T cells. Some thymocytes at the DP stage undergo signals to differentiate into natural killer T (NKT) cells. Gene expression analysis in thymocytes reveals that Bcl11b is upregulated at the transition from DN1 to DN2 and the expression is maintained in cells of αβ T cell lineages.
Immature DN thymocytes can be further divided into four subpopulations based on the surface expression of CD44 (or CD117/c-kit in case) and CD25, with the developmental progression being CD44+
(DN1) to CD44+
(DN2) to CD44−
(DN3) and then to CD44−
(DN4) cells (Fig. A). To make the developmental transition from DN3 to DP, proteins that are produced from productively rearranged genes at the TCRβ locus must be assembled into the preTCR complex and expressed on cell surface, which consists of a TCRβ-chain, the invariant pTα-chain, and CD3 components. At the DN4 stage, thymocytes re-enter into the cell cycle and rapidly proliferate.53,54)
Since the preTCR confers survival signals for DN3/DN4 cells, only cells that have acquired a functional preTCR can transit from DN3 to DP, a process known as β-selection. Thymocyte progenitor cells at the DN1/DN2 stage retain the capability to generate cells of non-T-cell lineages such as myeloid and natural killer (NK) cells.1,51)
However, DN3 thymocytes are committed T cells and have lost potentials to differentiate to other non-T-cell lineages.55,56)
Figure B shows flowcytomeric analysis of Bcl11bKO/KO
thymocytes using CD4/CD8 and CD25/CD44 markers. The Bcl11bKO/KO
thymus harbors DN and ISP cells but fail to produce DP thymocytes.3)
Further analysis showed that thymocytes at the DN3 stage retain normal cellularity but not at the DN4 stage, and the DN4 thymocytes exhibit apoptosis, accompanying low expression of anti-apoptotic proteins, Bcl-xL and Bcl-2.57)
As expected, Bcl11bKO/KO
DN4 thymocytes lack preTCR on the surface, though containing some within the cell. These indicate that developmental progression is impaired before the DP stage and probably around the DN3 stage where cells start to express the preTCR complex on cell surface. Bcl11bKO/KO
thymocytes exhibit less efficiency in DNA rearrangement of the TCRβ locus.57)
Recombination between D and J segments normally occurs while subsequent recombination between V and DJ segments is reduced, which could contribute in part to the lack of expression of preTCR complex. The lack of preTCR expression in DN4 thymocytes may be the cause of the apoptosis, which has been considered as a mechanism to eliminate deleterious cells within the thymus.
Loss of genes encoding a component of the preTCR complex in mice lacks the preTCR signaling and leads to the developmental arrest at DN3 stage As expected, introduction of the lost gene restores the ability to produce DP and SP cells.58–60)
In those mice, interestingly, deletion of the apoptosis-promoting gene p53
abrogates the developmental arrest to produce DP cells.61–63)
As for Bcl11bKO/KO
mice, introduction of functional TCR
β gene did not restore the developmental arrest.64)
This suggests that the preTCR complex formation alone cannot compensate the deficiency of Bcl11b. Furthermore, introduction of p53
deficiency into Bcl11bKO/KO
mice did not affect the developmental arrest and failed to inhibit apoptosis.64)
This suggests that p53-related apoptosis is not the reason for the failure of T-cell development upon loss of Bcl11b. These results suggest that Bcl11bKO/KO
thymocytes have defects in not only the pre-TCR signaling but some other signaling required for survival and transition to DP stage of development. Thus, the exact cause of T-cell defects in Bcl11b-lacking mutant mice remains unresolved.
Bcl11b also plays a role in the differentiation from DP to SP cells. This was demonstrated by analysis of CD4-Cre;Bcl11bflox/flox
mice, where loss of Bcl11b occurs in thymocytes after the DP stage.39,43)
The mice exhibited the developmental arrest at DP stage and did not produce SP cells and NKT cells. This indicates that Bcl11b is required for DP cells to differentiate to SP and NKT cells. The DP thymocytes underwent rearrangement at the TCRα gene normally but they failed to display proximal TCR signaling that is required for initiation of positive selection. Thus, the DP cells lacking TCR signaling underwent apoptosis during the process of positive selection. Interestingly, susceptibility to the apoptosis in those DP cells was at least in part independent of the anti-apoptotic factor Bcl2, because the introduction of Bcl2
transgene to CD4-Cre;Bcl11bflox/flox
mice did not fully prevent apoptosis of thymocytes.43)
These results indicate that Bcl11b plays critical roles in the establishment of TCR signaling in DP cells that is required for producing precursor cells of CD4 and CD8 lineages and also thymic NKT precursors.43)
Recently, three independent reports showed that deletion of Bcl11b blocks the progression from DN2 cells to DN3 committed T cells, indicating a role for Bcl11b at an early stage in T-cell development and maintenance of T-cell lineage commitment.22,46,47)
NK-like cells were generated in Bcl11bKO/KO
mice, and they may be converted from cells of T-cell lineage. This suggests that Bcl11b plays a role on the maintenance of T cell lineage identity in T cell lineage committed thymocytes, and its absence leads to reprogram T cells into the NK cell lineage. Hence, Bcl11b may regulate the cell fate choice between cells of T cell lineage and NK cells. One of the papers by Ikawa et al.
has succeeded in establishing a culture system that continuously cultures developmentally arrested and proliferating DN2 thymocytes in the presence of Delta-like 4 and the cytokine IL-7. Of importance, those DN2 thymocytes retain the potential to differentiate into multiple cell types, T cells, NK cells, dendritic cells, and macrophages. They discovered that the expression of Bcl11b in the proliferating DN2 cells leads to the relief of the differentiation arrest to differentiate into cells of T cell lineage. Also, they showed that simply reducing the concentration of IL-7 in the culture system stimulates robust T cell differentiation, suggesting that IL-7 controls the expression of Bcl11b and that Bcl11b is the critical T cell promoting transcription factor. Their study also includes the finding that identifies Bcl11b as a sensor that links cytokine signaling thresholds and T cell lineage commitment in early thymocyte progenitors.
As for CNS, expression of Bcl11b/Ctip2 was first detected in subcerebral projection neurons of the cerebral cortex, including developing corticospinal motor neurons (CSMN). Developmental analysis of Bcl11bKO/KO
mice showed defects in axonal extension and pathfinding by the projection neurons, resulting in failure of the neurons to connect to the spinal cord.4)
This indicates a critical role for Bcl11b in the development of corticospinal motor neurons. Interestingly, Bcl11bKO/+
heterozygous mice also show some subtle defects in CSMN fasciculation, suggesting haploinsufficiency of Bcl11b leading to phenotypic consequences. Further study showed expression of Bcl11b in GABAergic medium-sized spiny neurons (MSN) within the striatum that are derived from progenitors located in the germinal zone of the developing lateral ganglionic eminence.65)
Loss of Bcl11b function results in the failure of differentiation of MSN, leading to disruption of the patch-matrix organization of MSN. This suggests roles for Bcl11b in the differentiation of MSN and establishment of cellular architecture of the striatum.65)
Strial-enriched expression of Bcl11b was also demonstrated in adulthood,42)
suggesting that Bcl11b plays important roles in the functioning and maintenance of mature medium spiny neurons.
Recent study has shown that Bcl11b is also expressed in the developing vomeronasal system in the accessory olfactory bulb of the mouse as well.66)
The vomeronasal system detects pheromones to mediate social and reproductive behaviors in terrestrial vertebrates. In Bcl11bKO/KO
mice, vomeronasal sensory neurons (VSNs) are generated during development in the correct number but selectively die due to apoptosis. As a consequence, the mice display various phenotypes such as disorganization of layer formation of the accessory olfactory bulb, impaired axonal projections of VSNs, and defective mature differentiation of VSNs. The VSNs can be classified into two major types of neurons having different receptors. Interestingly, loss of Bcl11b function results in an impaired balance of cells of the two VSN types, suggesting that Bcl11b regulates the cell fate choice between the two different VSN types of neuronal cells.