The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability.

Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.

Wnt ligands from the embryonic surface ectoderm regulate 'bimetallic strip' optic cup morphogenesis in mouse.

The Wnt/ß-catenin response pathway is central to many developmental processes. Here, we assessed the role of Wnt signaling in early eye development using the mouse as a model system. We showed that the surface ectoderm region that includes the lens placode expressed 12 out of 19 possible Wnt ligands. When these activities were suppressed by conditional deletion of wntless (Le-cre; Wls(fl/fl)) there were dramatic consequences that included a saucer-shaped optic cup, ventral coloboma, and a deficiency of periocular mesenchyme. This phenotype shared features with that produced when the Wnt/ß-catenin pathway co-receptor Lrp6 is mutated or when retinoic acid (RA) signaling in the eye is compromised. Consistent with this, microarray and cell fate marker analysis identified a series of expression changes in genes known to be regulated by RA or by the Wnt/ß-catenin pathway. Using pathway reporters, we showed that Wnt ligands from the surface ectoderm directly or indirectly elicit a Wnt/ß-catenin response in retinal pigment epithelium (RPE) progenitors near the optic cup rim. In Le-cre; Wls(fl/fl) mice, the numbers of RPE cells are reduced and this can explain, using the principle of the bimetallic strip, the curvature of the optic cup. These data thus establish a novel hypothesis to explain how differential cell numbers in a bilayered epithelium can lead to shape change.

Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells.

Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.

Wntless functions in mature osteoblasts to regulate bone mass.

Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.

MICU1 regulates mitochondrial cristae structure and function independently of the mitochondrial Ca2+ uniporter channel.

MICU1 is a calcium (Ca2+)-binding protein that regulates the mitochondrial Ca2+ uniporter channel complex (mtCU) and mitochondrial Ca2+ uptake. MICU1 knockout mice display disorganized mitochondrial architecture, a phenotype that is distinct from that of mice with deficiencies in other mtCU subunits and, thus, is likely not explained by changes in mitochondrial matrix Ca2+ content. Using proteomic and cellular imaging techniques, we found that MICU1 localized to the mitochondrial contact site and cristae organizing system (MICOS) and directly interacted with the MICOS components MIC60 and CHCHD2 independently of the mtCU. We demonstrated that MICU1 was essential for MICOS complex formation and that MICU1 ablation resulted in altered cristae organization, mitochondrial ultrastructure, mitochondrial membrane dynamics, and cell death signaling. Together, our results suggest that MICU1 is an intermembrane space Ca2+ sensor that modulates mitochondrial membrane dynamics independently of matrix Ca2+ uptake. This system enables distinct Ca2+ signaling in the mitochondrial matrix and at the intermembrane space to modulate cellular energetics and cell death in a concerted manner.

CD11c(hi) dendritic cells regulate the re-establishment of vascular quiescence and stabilization after immune stimulation of lymph nodes.

Lymph node expansion during immune responses is accompanied by rapid vascular expansion. The re-establishment of quiescence and stabilization of the newly expanded vasculature and the regulatory mechanisms involved have not been well studied. We show that although initiation of vascular expansion in immune-stimulated nodes is associated with upregulated endothelial cell proliferation, increased high endothelial venule trafficking efficiency and VCAM-1 expression, and disrupted perivascular fibroblastic reticular cell organization, the re-establishment of vascular quiescence and stabilization postexpansion is characterized by reversal of these phenomena. Although CD11c(med) cells are associated with the initiation of vascular expansion, CD11c(hi)MHC class II (MHC II)(med) dendritic cells (DCs) accumulate later, and their short-term depletion in mice abrogates the re-establishment of vascular quiescence and stabilization. CD11c(hi)MHC II(med) cells promote endothelial cell quiescence in vitro and, in vivo, mediate quiescence at least in part by mediating reduced lymph node vascular endothelial growth factor. Disrupted vascular quiescence and stabilization in expanded nodes is associated with attenuated T cell-dependent B cell responses. These results describe a novel mechanism whereby CD11c(hi)MHC II(med) DCs regulate the re-establishment of vascular quiescence and stabilization after lymph node vascular expansion and suggest that these DCs function in part to orchestrate the microenvironmental alterations required for successful immunity.

Generation of mice with a conditional null allele for Wntless.

The Wnt-signaling pathway is necessary in a variety of developmental processes and has been implicated in numerous pathologies. Wntless (Wls) binds to Wnt proteins and facilitates Wnt sorting and secretion. Conventional deletion of Wls results in early fetal lethality due to defects in body axis establishment. To gain insight into the function of Wls in later stages of development, we have generated a conditional null allele. Homozygous germline deletion of Wls confirmed prenatal lethality and failure of embryonic axis formation. Deletion of Wls using Wnt1-cre phenocopied Wnt1 null abnormalities in the midbrain and hindbrain. In addition, conditional deletion of Wls in pancreatic precursor cells resulted in pancreatic hypoplasia similar to that previously observed after conditional ß-catenin deletion. This Wls conditional null allele will be valuable in detecting novel Wnt functions in development and disease.

Fibroblast-type reticular stromal cells regulate the lymph node vasculature.

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)(+) endothelial cells are less sensitive than PNAd(-) endothelial cells to VEGF blockade. Lymphotoxin beta receptor (LTbetaR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTbetaR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTbetaR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd(+) and PNAd(-) endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.