Functional requirement of CCN2 for intramembranous bone formation in embryonic mice

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and Ccn2 null mutant mice display skeletal dysmorphisms. However, little is known concerning the roles of CCN2 during bone formation. We herein present a comparative analysis of wild-type and Ccn2 null mice to investigate the roles of CCN2 in bone development. Multiple histochemical methods were employed to analyze the effects of CCN2 deletion in vivo, and effects of CCN2 on the osteogenic response were evaluated with the isolated and cultured osteoblasts. As a result, we found a drastic reduction of the osteoblastic phenotype in Ccn2 null mutants. Importantly, addition of exogenous CCN2 promoted every step of osteoblast differentiation and rescued the attenuated activities of the Ccn2 null osteoblasts. These results suggest that CCN2 is required not only for the regulation of cartilage and subsequent events, but also for the normal intramembranous bone development.     

Conditional deletions refine the embryonic requirement for Dlk1

Numerous studies have implicated Delta-like 1 (DLK1), a transmembrane protein that shares homology with Notch ligands, in embryonic growth and differentiation. Dlk1 expression is widespread, though not ubiquitous, during early development, but is confined to a few specific cell types in adults. Adult Dlk1-expressing tissues include the Insulin-producing β-cells of the pancreas and the Growth hormone-producing somatotrophs of the pituitary gland. Previously generated Dlk1 null mice (Dlk1^Sul-pat), display a partially penetrant neonatal lethality and a complex pattern of developmental and adult phenotypes. Here we describe the generation of a conditional Dlk1 mouse line (Dlk1^flox) to facilitate cell type-specific deletion of the Dlk1 gene, providing a powerful system to explore each aspect of the Dlk1 null phenotype. Four tissue-specific Cre mouse lines were used to produce individual Dlk1 deletions in pancreatic β-cells, pituitary somatotrophs and the endothelial cells of the embryo and placenta, key candidates for the Dlk1 phenotype. Contrary to expectations, all of these conditional mice were fully viable, and none recapitulated any aspect of the Dlk1^Sul-pat null mice. Dlk1 expression is therefore not essential for the normal development of β-cells, somatotrophs and endothelial cells, and the tissues responsible for the Dlk1 null phenotype remain to be identified. Dlk1^flox mice will continue to provide an important tool for further research into the function of Dlk1.     

Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins.

Neuropilin-1 (np1) and neuropilin-2 (np2) are receptors for class-3 semaphorins and for several isoforms of VEGF. We have cloned and characterized two chick isoforms of np2 cDNA. Expression patterns of np1, np2, and ephrin-B2 were compared in the developing vascular system of 24-72 h old chick embryos. We show for the first time that np2 is expressed in blood vessels in vivo from the earliest stages of their formation. In contrast to ephrin-B2, both np1 and np2 are expressed in blood islands of 24 h old chick embryos. At 48-72 h, np1 expression is localized preferentially in arteries with an expression pattern that resembles that of ephrin-B2. In contrast, np2 is expressed preferentially in veins. Thus, neuropilins may play a role in determining the arterial or venous identity of blood vessels.     

A requirement for reversible binding between aggregating embryonic cells before stable adhesion.

Chick embryonic liver and neural retina cells aggregate in a two-step process. Initially, cells formed a loose association in a step that apparently did not require metabolic energy. Cells bound in this manner were dissociable by mild shear forces or by simple dilution. The results of the dilution experiments suggest a readily reversible binding of single cells to form these types of aggregates. In a second step, which required metabolic energy, the cells became firmly, or stably attached. The formation of both types of bond was temperature-dependent. Kinetic studies indicated that the formation of reversible bonds between cells was required before the cells could become stably attached, and that reversibly bound cells were converted directly into stably bound cells.     

Blood vessel formation in cerebral organoids formed from human embryonic stem cells

Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.     

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17⁺ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17⁺ cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP⁺ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17⁺ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.     

Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus.

The nickel-dependent chemolithoautotrophic growth of Alcaligenes eutrophus is apparently due to a requirement of nickel for active hydrogenase formation. Cells grown heterotrophically with fructose and glycerol revealed a specific activity of soluble and membrane-bound hydrogenase which was severalfold higher than the normal autotrophic level. The omission of nickel from the medium did not affect heterotrophic growth, but the soluble hydrogenase activity was reduced significantly. In the presence of ethylenediaminetetraacetic acid (EDTA), almost no hydrogenase activity was detected. The addition of nickel allowed active hydrogenase formation even when EDTA was present. When chloramphenicol was added simultaneously with nickel to an EDTA-containing medium, almost no hydrogenase activity was found. This indicates that nickel ions are involved in a process which requires protein synthesis and not the direct reactivation of a preformed inactive protein. The formation of the membrane-bound hydrogenase also appeared to be nickel dependent. Autotrophic CO2 assimilation did not specifically require nickel ions, since formate was utilized in the presence of EDTA and the activity of ribulosebisphosphate carboxylase was not affected under these conditions.     

A requirement for NF-protocadherin and TAF1/Set in cell adhesion and neural tube formation

Neurulation in vertebrates is an intricate process requiring extensive alterations in cell contacts and cellular morphologies as the cells in the neural ectoderm shape and form the neural folds and neural tube. Despite these complex interactions, little is known concerning the molecules that mediate cell adhesion within the embryonic neural plate and neural folds. Here, we demonstrate the requirement for NF-protocadherin (NFPC) and its cytosolic partner TAF1/Set for proper neurulation in Xenopus. Both NFPC and TAF1 function in cell-cell adhesion in the neural ectoderm, and disruptions in either NFPC or TAF1 result in a failure of the neural tube to close. This neural tube defect can be attributed to a lack of proper organization of the cells in the dorsal neural folds, manifested by a loss in the columnar epithelial morphology and apical localization of F-actin. However, the epidermal ectoderm is still able to migrate and cover the open neural tube, indicating that the fusions of the neural tube and epidermis are separate events. These studies demonstrate that NFPC and TAF1 function to maintain proper cell-cell interactions within the neural folds and suggest that NFPC and TAF1 participate in novel adhesive mechanisms that contribute to the final events of vertebrate neurulation.     

Immunoglobulin carbohydrate requirement for formation of an IgG-IgG complex.

In addition to their Fc oligosaccharides, some immunoglobulin molecules have oligosaccharides linked to variable segments of H or L chains. These Fab oligosaccharides are potential determinants of antibody specificity. This possibility was considered in a study of the IgG antiglobulin from a patient with IgG-IgG complexes. F(ab')2 fragments of the antiglobulin retained the ability to form complexes with normal IgG as detected by analytical ultracentrifugation. Removal of F(ab')2 sialic acids by neuraminidase abolished complex formation. Recombination experiments further localized antiglobulin activity to the L chains. Antiglobulin activity of the recombinant molecules was shown by analytical ultracentrifugation and by column chromatography with molecules containing 125I-labeled L chains. L chains from the subject's IgG were enriched in sialic acids. Thus, a sialic acid-containing oligosaccharide on the L chain of this antiglobulin is required for its binding action.     

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

BMP4 and noggin control embryonic blood vessel formation by antagonistic regulation of VEGFR-2 (Quek1) expression

Regulation of VEGFR-2 (Quek1) is an important mechanism during blood vessel formation. In the paraxial mesoderm, Quek1 expression is restricted to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube. By grafting of either intermediate mesoderm or BMP4 beads into the paraxial mesoderm, we show that BMP4 is a positive regulator of VEGFR-2 (Quek1) expression in the quail embryo. Separation of somites from intermediate mesoderm leads to down-regulation of Quek1 expression. The expression of Quek1 in the medial somite half is normally repressed by the notochord and becomes up-regulated and lateromedially expanded after separation of the notochord. Our results show that up-regulation of BMP4 leads to an increase of the number of blood vessels, whereas inhibition of BMP4 by noggin results in a reduction of blood vessels.     

Two-factor requirement for murine megakaryocyte colony formation

WEHI-3 cell-conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G-150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI-3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum-free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.