Glenn L. Radice

Lack of Evidence that Hematopoietic Stem Cells Depend on N-Cadherin-Mediated Adhesion to Osteoblasts for Their Maintenance

Recent studies have proposed that bone marrow hematopoietic stem cells (HSCs) are maintained via N-cadherin-mediated homophilic adhesion with osteoblasts. However, there is not yet any evidence that N-cadherin-expressing cells have HSC activity or that osteoblasts are required for HSC maintenance. We were unable to detect N-cadherin expression in highly purified HSCs by polymerase chain reaction, by using commercial anti-N-cadherin antibodies, or by beta-galactosidase staining of N-cadherin gene trap mice. Only N-cadherin-negative bone marrow cells exhibited HSC activity in irradiated mice. Finally, biglycan-deficient mice had significant reductions in trabecular bone and osteoblasts but showed no defects in hematopoiesis, HSC frequency, or function. Thus, reductions in osteoblasts do not necessarily lead to reductions in HSCs. Most bone marrow HSCs in wild-type and biglycan-deficient mice localized to sinusoids, and few localized within five cell diameters of the endosteum. These results question whether significant numbers of HSCs depend on N-cadherin-mediated adhesion to osteoblasts.

Induced Deletion of the N-Cadherin Gene in the Heart Leads to Dissolution of the Intercalated Disc Structure

The structural integrity of the heart is maintained by the end-to-end connection between the myocytes called the intercalated disc. The intercalated disc contains different junctional complexes that enable the myocardium to function as a syncytium. One of the junctional complexes, the zonula adherens or adherens junction, consists of the cell adhesion molecule, N-cadherin, which mediates strong homophilic cell–cell adhesion via linkage to the actin cytoskeleton. To determine the function of N-cadherin in the working myocardium, we generated a conditional knockout containing loxP sites flanking exon 1 of the N-cadherin (Cdh2) gene. Using a cardiac-specific tamoxifen-inducible Cre transgene, N-cadherin was deleted in the adult myocardium. Loss of N-cadherin resulted in disassembly of the intercalated disc structure, including adherens junctions and desmosomes. The mutant mice exhibited modest dilated cardiomyopathy and impaired cardiac function, with most animals dying within two months after tamoxifen administration. Decreased sarcomere length and increased Z-line thickness were observed in the mutant hearts consistent with loss of muscle tension because N-cadherin was no longer available to anchor myofibrils at the plasma membrane. Ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous ventricular tachycardia, confirming that the deaths were arrhythmic in nature. A significant decrease in the gap junction protein, connexin 43, was observed in the N-cadherin–depleted hearts. This animal model provides the first demonstration of the hierarchical relationship of the structural components of the intercalated disc in the working myocardium, thus establishing N-cadherin’s paramount importance in maintaining the structural integrity of the heart.

Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.

Connexin 43 (Cx43α1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43α1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin–deficient neural crest cells, but the alterations were different from that elicited by Cx43α1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43α1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43α1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43α1 deficiency. Based on these findings, we propose a model in which Cx43α1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cells locomotory apparatus through p120ctn signaling.

Male Infertility, Impaired Sperm Motility, and Hydrocephalus in Mice Deficient in Sperm-Associated Antigen 6

ABSTRACT Gene targeting was used to create mice lacking sperm-associated antigen 6 (Spag6), the murine orthologue of Chlamydomonas PF16, an axonemal protein containing eight armadillo repeats predicted to be important for flagellar motility and stability of the axoneme central apparatus. Within 8 weeks of birth, approximately 50% of Spag6-deficient animals died with hydrocephalus. Spag6-deficient males surviving to maturity were infertile. Their sperm had marked motility defects and was morphologically abnormal with frequent loss of the sperm head and disorganization of flagellar structures, including loss of the central pair of microtubules and disorganization of the outer dense fibers and fibrous sheath. We conclude that Spag6 is essential for sperm flagellar motility and that it is important for the maintenance of the structural integrity of mature sperm. The occurrence of hydrocephalus in the mutant mice also implicates Spag6 in the motility of ependymal cilia.

Cardiac-Specific Loss of N-Cadherin Leads to Alteration in Connexins With Conduction Slowing and Arrhythmogenesis

The remodeling of ventricular gap junctions, as defined by changes in size, distribution, or function, is a prominent feature of diseased myocardium. However, the regulation of assembly and maintenance of gap junctions remains poorly understood. To investigate N-cadherin function in the adult myocardium, we used a floxed N-cadherin gene in conjunction with a cardiac-specific tamoxifen-inducible Cre transgene. The mutant animals appeared active and healthy until their sudden death ≈2 months after deleting N-cadherin from the heart. Electrophysiologic analysis revealed abnormal conduction in the ventricles of mutant animals, including diminished QRS complex amplitude consistent with loss of electrical coupling in the myocardium. A significant decrease in the gap junction proteins, connexin-43 and connexin-40, was observed in N-cadherin–depleted myocytes. Perturbation of connexin function resulted in decreased ventricular conduction velocity, as determined by optical mapping. Our data suggest that perturbation of the N-cadherin/catenin complex in heart disease may be an underlying cause, leading to the establishment of the arrythmogenic substrate by destabilizing gap junctions at the cell surface.

N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis

Endothelial cells express two classic cadherins, VE-cadherin and N-cadherin. The importance of VE-cadherin in vascular development is well known; however, the function of N-cadherin in endothelial cells remains poorly understood. Contrary to previous studies, we found that N-cadherin localizes to endothelial cell–cell junctions in addition to its well-known diffusive membrane expression. To investigate the role of N-cadherin in vascular development, N-cadherin was specifically deleted from endothelial cells in mice. Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects. Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn. The down-regulation of both VE-cadherin and p120ctn was confirmed in cultured endothelial cells using small interfering RNA to knockdown N-cadherin. We also show that N-cadherin is important for endothelial cell proliferation and motility. These findings provide a novel paradigm by which N-cadherin regulates angiogenesis, in part, by controlling VE-cadherin expression at the cell membrane.

N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.

Adhesive and Signaling Functions of Cadherins and Catenins in Vertebrate Development

Properly regulated intercellular adhesion is critical for normal development of all metazoan organisms. Adherens junctions play an especially prominent role in development because they link the adhesive function of cadherin-catenin protein complexes to the dynamic forces of the actin cytoskeleton, which helps to orchestrate a spatially confined and very dynamic assembly of intercellular connections. Intriguingly, in addition to maintaining intercellular adhesion, cadherin-catenin proteins are linked to several major developmental signaling pathways crucial for normal morphogenesis. In this article we will highlight the key genetic studies that uncovered the role of cadherin-catenin proteins in vertebrate development and discuss the potential role of these proteins as molecular biosensors of external cellular microenvironment that may spatially confine signaling molecules and polarity cues to orchestrate cellular behavior throughout the complex process of normal morphogenesis.

N-Cadherin-Mediated Human Granulosa Cell Adhesion Prevents Apoptosis : A Role in Follicular Atresia and Luteolysis?

Studies suggest that cell-cell interactions may regulate apoptosis, and in particular, the calcium-dependent cell adhesion molecule N-cadherin has been shown to be capable of modulating this process. Rat granulosa cells (GCs) are known to express N-cadherin whereas cAMP is known to induce apoptosis in human and rat GCs. Based on these observations, we hypothesized that N-cadherin regulates human GC apoptosis via a cAMP-dependent mechanism. N-cadherin expression was evaluated in ovarian follicles and corpora lutea utilizing immunohistochemical techniques and in luteinized GCs in culture using immunoblotting, flow cytometric analysis, immunohistochemistry, and indirect immunofluorescence techniques utilizing anti-N-cadherin antibodies directed against both the extracellular and cytoplasmic domains of the molecule. Apoptosis was assessed by TUNEL and DNA fragmentation analysis and confirmed by flow cytometric cell cycle analysis and electron microscopy. The rate of GC apoptosis was found to be two- to three-fold lower among aggregated cells, as compared with single cells. N-cadherin was found to be expressed by aggregating GCs in vitro and GCs cultured in the presence of either N-cadherin function disrupting antibodies or peptides exhibiting enhanced rates of apoptosis. GCs in situ stained intensely for N-cadherin in preantral and normal growing preovulatory follicles as well as early corpora lutea. N-cadherin was weak in atretic follicles and regressing corpora lutea. Exposure of GCs to cAMP increased apoptosis while decreasing N-cadherin protein expression in a dose-dependent manner. Cell culture under serum-free conditions increased apoptosis and decreased N-cadherin expression, in part through cleavage of the extracellular domain of the molecule. The metalloproteinase inhibitor 1-10-phenanthroline inhibited the cleavage of the extracellular domain of N-cadherin and concomitantly inhibited the serum-deprivation-induced apoptosis of aggregated GCs. Collectively, these observations suggest that down-regulation of N-cadherin or the absence of a functional extracellular domain of the molecule prevents cell aggregation and is associated with GC apoptosis. In addition, cAMP induces apoptosis in a dose-dependent manner, and this process is dependent, at least in part, on regulation of the N-cadherin molecule at the surface of the cells. We conclude that N-cadherin-mediated GC signaling plays a central role in follicular and luteal cell survival.

Co-operative roles for E-cadherin and N-cadherin during lens vesicle separation and lens epithelial cell survival.

The classical cadherins are known to have both adhesive and signaling functions. It has also been proposed that localized regulation of cadherin activity may be important in cell assortment during development. In the context of eye development, it has been suggested that cadherins are important for separation of the invaginated lens vesicle from the surface ectoderm. To test this hypothesis, we conditionally deleted N-cadherin or E-cadherin from the presumptive lens ectoderm of the mouse. Conditional deletion of either cadherin alone did not produce a lens vesicle separation defect. However, these conditional mutants did exhibit common structural deficits, including microphthalmia, severe iris hyperplasia, persistent vacuolization within the fibre cell region, and eventual lens epithelial cell deterioration. To assess the co-operative roles of E-cadherin and N-cadherin within the developing lens, double conditional knockout embryos were generated. These mice displayed distinct defects in lens vesicle separation and persistent expression of another classical cadherin, P-cadherin, within the cells of the persistent lens stalk. Double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival. Finally, the severity of the lens phenotype was shown to be sensitive to the number of wild-type E- and N-cadherin alleles. These data suggest that the co-operative expression of both E- and N-cadherin during lens development is essential for normal cell sorting and subsequent lens vesicle separation.