Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

Regulation of basal body and ciliary functions by Diversin

The centrosome is essential for the formation of the cilia and has been implicated in cell polarization and signaling during early embryonic development. A number of Wnt pathway components were found to localize at the centrosome, but how this localization relates to their signaling functions is unclear. In this study, we assessed a role for Diversin, a putative Wnt pathway mediator, in developmental processes that involve cilia. We find that Diversin is specifically localized to the basal body compartment near the base of the cilium in Xenopus multi-ciliated skin cells. Overexpression of Diversin RNA disrupted basal body polarization in these cells, suggesting that tightly regulated control of Diversin levels is crucial for this process. In cells depleted of endogenous Diversin, basal body structure appeared abnormal and this was accompanied by disrupted polarity, shortened or absent cilia and defective ciliary flow. These results are consistent with the involvement of Diversin in processes that are related to the acquisition of cell polarity and require ciliary functions.     

Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice

Renin plays a key role in controlling blood pressure through its specific cleavage of angiotensinogen to generate angiotensin I (AI). Although possible existence of the other angiotensin forming enzymes has been discussed to date, its in vivo function remains to be elucidated. To address the contribution of renin, we generated renin knockout mice. Homozygous mutant mice show neither detectable levels of plasma renin activity nor plasma AI, lowered blood pressure 20-30 mm Hg less than normal, increased urine and drinking volume, and altered renal morphology as those observed in angiotensinogen-deficient mice. We recently found the decreased density in granular layer cells of hippocampus and the impaired blood-brain barrier function in angiotensinogen-deficient mice. Surprisingly, however, such brain phenotypes were not observed in renin-deficient mice. Our results demonstrate an indispensable role for renin in the circulating angiotensin generation and in the maintenance of blood pressure, but suggest a dispensable role for renin in the blood-brain barrier function.     

Three-dimensional structure of basal body triplet revealed by electron cryo-tomography

Basal bodies and centrioles play central roles in microtubule (MT)‐organizing centres within many eukaryotes. They share a barrel‐shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo‐tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo‐atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non‐tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ‐tubulin and other components participate in the assembly of the basal body.     

Coordination of Golgi functions by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI4Ks) regulate vesicle-mediated export from the Golgi apparatus via phosphatidylinositol 4-phosphate (PtdIns4P) binding effector proteins that control vesicle budding reactions and regulate membrane dynamics. Evidence has emerged from the characterization of Golgi PI4K effectors that vesicle budding and lipid dynamics are tightly coupled via a regulatory network that ensures that the appropriate membrane composition is established before a transport vesicle buds from the Golgi. An important hub of this network is protein kinase D, which regulates the activity of PI4K and several PtdIns4P effectors that control sphingolipid and sterol content of Golgi membranes. Other newly identified PtdIns4P effectors include Vps74/GOLPH3, a phospholipid flippase called Drs2 and Sec2, a Rab guanine nucleotide exchange factor (GEF). These effectors orchestrate membrane transformation events facilitating vesicle formation and targeting. In this review, we discuss how PtdIns4P signaling is integrated with membrane biosynthetic and vesicle budding machineries to potentially coordinate these crucial functions of the Golgi apparatus.     

Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction

Treatment of chick embryo fibroblasts with 0.5% Triton® X-100 extracts most of the cell protein, leaving an organized part of the cell structure attached to the tissue culture dish. This “Triton cytoskeleton” consists largely of intermediate-sized filaments and bundles of microfilaments. SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton is made up of three main proteins. One protein component is 42,000 daltons and co-migrates with muscle actin. The other two components are 52,000 and 230,000 daltons and remain quantitatively associated with the cytoskeleton during the detergent extraction. The possible identity of these three protein components and their organization into a supramolecular structure is discussed.     

白血病抑制因子及其在胚胎发育和胚泡着床中的生理功能

白血病抑制因子在哺乳动物妊娠早期中具有重要的生物学功能。胚源和母体白血病抑制因子可作为胚胎营养因子促进胚胎的发育;母体白血病抑制因子可能通过调节子宫接受性和／或激活胚泡从而启动胚泡着床。     

Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP

The CaMV 35S promoter is the most commonly used promoter for driving transgene expression in plants. Though it is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. In addition, the information available on its expression profile in all possible cell and tissue types and during early stages of development is incomplete. We present here a detailed expression profile of this promoter investigated using the green fluorescent protein (GFP) gene as a reporter system in cotton during embryo development, and in all the vegetative and floral cell and tissue types. GFP expression was not detected during the early stages of embryogenesis. The first perceptible GFP expression was observed in a small area at the junction of hypocotyl and cotyledons in embryos at around 13 days after anthesis. The GFP fluorescence progressively became stronger and expanded throughout the cotyledon and hypocotyl as embryo development advanced. After germination, varying levels of promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf, petiole, and root. The promoter was also expressed in all floral parts. Although cotton pollen exhibited a low level of greenish autofluorescence, it was possible to discern GFP-dependent fluorescence in some of the pollen from all the T₀ plants examined. Developing cotton fibers also exhibited GFP fluorescence suggesting that the 35S promoter was active in these specialized epidermal cells. Thus, we show that the expression of the 35S promoter was developmentally regulated during embryogenesis and that beyond a certain stage during embryogenesis, the promoter was expressed in most cell and tissue types in cotton albeit at different levels.     

Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis

Arrestin binding to activated, phosphorylated G protein-coupled receptors (GPCRs) represents a critical step in regulation of light- and hormone-dependent signaling. Nonvisual arrestins, such as arrestin-2, interact with multiple proteins for the purpose of propagating and terminating signaling events. Using a combination of X-ray crystallography, molecular modeling, mutagenesis, and binding analysis, we reveal structural features of arrestin-2 that may enable simultaneous binding to phosphorylated receptor, SH3 domains, phosphoinositides, and beta-adaptin. The structure of full-length arrestin-2 thus provides a uniquely oriented scaffold for assembly of multiple, diverse molecules involved in GPCR signal transduction.     

Regional and developmental characteristics of human embryo mosaicism revealed by single cell sequencing

Chromosomal mosaicism is common throughout human pre- and post-implantation development. However, the incidence and characteristics of mosaicism in human blastocyst remain unclear. Concerns and confusions still exist regarding the interpretation of chromosomal mosaicism on preimplantation genetic testing for aneuploidy (PGT-A) results and embryo development. Here, we aimed to estimate the genetic concordance between trophectoderm (TE), inner cell mass (ICM) and the corresponding human embryonic stem cells (hESCs), and to explore the characteristics of mosaicism in human blastocyst and hESCs on a single cell level. The single cell sequencing results of TE cells indicated that 65.71% of the blastocysts were mosaic (23 in 35 embryos), while the ICM sequencing results suggested that 60.00% of the blastocysts were mosaic (9 in 15 embryos). The incidence of mosaicism for the corresponding hESCs was 33.33% (2 in 6 embryos). No significant difference was observed between the mosaic rate of TE and that of ICM. However, the mosaic rate of the corresponding hESCs was significantly lower than that of TE and ICM cells, suggesting that the incidence of mosaicism may decline during embryonic development. Upon single cell sequencing, we found several “complementary” copy number variations (CNVs) that were usually not revealed in clinical PGT-A which used multi-cell DNA sequencing (or array analysis). This indicates the potential diagnostic risk of PGT-A based multi-cell analysis routinely in clinical practice. This study provided new insights into the characteristics, and considerable influences, of mosaicism on human embryo development, as well as the clinical risks of PGT-A based on multi-cell biopsies and bulk DNA assays.     

Essential roles of carbohydrate signals in development, immune response and tissue functions, as revealed by gene targeting

Knockout mice lacking glycosyltransferases or sulfotransferases provide unequivocal evidence that the carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans play essential roles in various biological phenomena such as development, the immune response, and tissue functions. Examples of abnormalities of null mutants include arrest of embryogenesis due to deletion of N-acetylglucosaminyltransferase I or glucosylceramide synthase, failure of kidney formation in heparan sulfate 2-O-sulfotransferase deficiency, suppressed antibody production in alpha-2, 6-sialyltransferase deficiency, male sterility in GM2/GD2 synthase deficiency, and abnormalities in the function and stability of myelin in galactosylceramide deficiency.