Id4 is required for the correct timing of neural differentiation

Complex intrinsic and extrinsic mechanisms determine neural cell fate during development of the nervous system. Using Id4 deficient mice, we show that Id4 is required for normal development of the central nervous system (CNS), timing neural differentiation in the developing forebrain. In the absence of Id4, the ventricular zone of the neocortex, future hippocampus as well as lateral and medial ganglionic eminences exhibited a 20-30% reduction in mitotic neural precursor cells (NPCs). Although the number of apoptotic cells was significantly increased, the neocortex of Id4(-/-) embryos was consistently thicker due to premature neuronal differentiation, which resulted in an increase in early-born neurons in the adult Id4(-/-) cortex. Late-born cortical neurons and astrocytes in the cortex, septum, hippocampus and caudate putamen of Id4(-/-) adult brains were decreased, however, likely due to the depletion of the NPC pool. Consequently, adult Id4(-/-) brains were smaller and exhibited enlarged ventricles. In vitro analysis of neurosphere cultures revealed that proliferation of Id4-deficient NPCs was impaired and that BMP2-mediated astrocyte differentiation was accelerated in the absence of Id4. Together, these in vivo and in vitro data suggest a crucial role for Id4 in regulating NPC proliferation and differentiation.     

Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells

Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and play an important role in development and in many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitors of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation. We demonstrated that the expression of Id-1, Id-2, and Id-3 genes was significantly induced at the early stage of BMP-9 stimulation and returned to basal levels at 3 days after stimulation. RNA interference-mediated knockdown of Id expression significantly diminished the BMP-9-induced osteogenic differentiation of mesenchymal progenitor cells. Surprisingly, a constitutive overexpression of these Id genes also inhibited osteoblast differentiation initiated by BMP-9. Furthermore, we demonstrated that BMP-9-regulated Id expression is Smad4-dependent. Overexpression of the three Id genes was shown to promote cell proliferation that was coupled with an inhibition of osteogenic differentiation. Thus, our findings suggest that the Id helix-loop-helix proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells and that Id expression must be down-regulated during the terminal differentiation of committed osteoblasts, suggesting that a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells.     

Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus

T cell differentiation in the thymus depends on sequential interactions between lymphoid progenitors and stromal cells in discrete regions of the cortex. Here we show that CXCL12/CXCR4 signaling is absolutely required for proper localization of early progenitors into the cortex and thus for successful steady state differentiation. All early progenitors in the thymus express CXCR4, and its ligand (CXCL12) is expressed only by stromal cells in the cortex, where early progenitors are found. Early progenitors migrate in response to CXCL12 in vitro, while thymus-specific deletion of CXCR4 in vivo results in failed cortical localization and developmental arrest. These findings indicate a crucial and nonredundant role for CXCR4 in facilitating localization of early lymphoid progenitors to tissue regions of the thymus, where lineage commitment and proliferation are controlled.     

ID4 regulates mammary gland development by suppressing p38MAPK activity

The ID family of helix-loop-helix proteins regulates cell proliferation and differentiation in many different developmental pathways, but the functions of ID4 in mammary development are unknown. We report that mouse Id4 is expressed in cap cells, basal cells and in a subset of luminal epithelial cells, and that its targeted deletion impairs ductal expansion and branching morphogenesis as well as cell proliferation induced by estrogen and/or progesterone. We discover that p38MAPK is activated in Id4-null mammary cells. p38MAPK is also activated following siRNA-mediated Id4 knockdown in transformed mammary cells. This p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands. Therefore, ID4 promotes mammary gland development by suppressing p38MAPK activity.     

Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ(1)

Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.     

Cell Proliferation,Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex

Appropriate maintenance and regeneration of adult endocrine organs is important in both normal physiology and disease. We investigated cell proliferation, movement and differentiation in the adult mouse adrenal cortex, using different 5-bromo-2''-deoxyuridine (BrdU) labelling regimens and immunostaining for phenotypic steroidogenic cell markers. Pulse-labelling showed that cell division was largely confined to the outer cortex, with most cells moving inwards towards the medulla at around 13-20 µm per day, though a distinct labelled cell population remained in the outer 10% of the cortex. Pulse-chase-labelling coupled with phenotypic immunostaining showed that, unlike cells in the inner cortex, most BrdU-positive outer cortical cells did not express steroidogenic markers, while co-staining for BrdU and Ki67 revealed that some outer cortical BrdU-positive cells were induced to proliferate following acute adrenocorticotropic hormone (ACTH) treatment. Extended pulse-chase-labelling identified cells in the outer cortex which retained BrdU label for up to 18-23 weeks. Together, these observations are consistent with the location of both slow-cycling stem/progenitor and transiently amplifying cell populations in the outer cortex. Understanding the relationships between these distinct adrenocortical cell populations will be crucial to clarify mechanisms underpinning adrenocortical maintenance and long-term adaptation to pathophysiological states.     

Lactation defect in mice lacking the helix-loop-helix inhibitor Id2

Id proteins are thought to be negative regulators of cell differentiation and positive regulators of cell proliferation. Mammary glands of Id2(-/-) female mice reveal severely impaired lobulo-alveolar development during pregnancy. Id2(-/-) mammary epithelia show no precocious maturation, but instead exhibit intrinsic defects in both cell proliferation and cell survival, implying that the role of Id2 in pregnant mammary epithelia is mainly stimulation of cell proliferation and support of cell viability. Expression studies of genes required for mammary gland development suggest Id2 to be a downstream or parallel factor of these genes. A decrease in the DNA binding activity of Stat5 was also observed in Id2(-/-) mammary glands at 7 days post-coitus. Our results indicate an indispensable role of Id2 in pregnant mammary glands.     

Id2a influences neuron and glia formation in the zebrafish retina by modulating retinoblast cell cycle kinetics

Inhibitor of differentiation (Id) family helix-loop-helix proteins regulate the proliferation, survival and differentiation of numerous cell types during development; however, their functions during retinal development have not been analyzed. Using loss-of-function and overexpression assays in zebrafish, we demonstrate that Id2a levels modulate retinoblast cell cycle kinetics and thereby influence neuron and glia formation in the retina. Id2a-deficient retinas possess increased numbers of cells occupying S phase, at the expense of mitotic cells, and kinetic analyses demonstrate that Id2a is required for S-phase progression and/or the transition from S to M phase. Id2a-dependent defects in retinoblast proliferation lead to microphthalmia and to an absence of nearly all differentiated inner and outer nuclear layer cell types. Overexpression of id2a has the opposite effect on retinoblast cell cycle kinetics: id2a-overexpressing retinoblasts progress from S to M phase more rapidly and they undergo mitosis more frequently, which results in macrophthalmia. Mosaic analyses reveal that Id2a function in facilitating both cell cycle progression and neuronal differentiation in the retina is non-cell-autonomous, suggesting that Id2a functions upstream of the extrinsic pathways that regulate retinogenesis.