Scanning electron microscope study of the developing trout pelvic fin bud

The growth of the pelvic fin bud has been studied with the SEM along with the characteristics of the pseudoapical epidermal ridge which occupies the free margin of the bud. SEM revealed fluffy protuberances in many of the epidermal cells, distinguishing the fin bud territory from adjacent areas. When the pseudoapical ridge appears, all the cells show this feature but their relative number decreases and these cells, termed the "tassel cells," are finally restricted to the base of the fin bud. This particular surface structure of the superficial cells may be unique to the fish, since it has not been heretofore reported in SEM studies of tetrapod limb bud.     

Comprehensive and quantitative analysis of yeast deletion mutants defective in apical and isotropic bud growth

To obtain a comprehensive understanding of the budding phase transition, 4,711 Saccharomyces cerevisiae haploid nonessential gene deletion mutants were screened with the image processing program CalMorph, and 35 mutants with a round bud and 173 mutants with an elongated bud were statistically identified. We classified round and elongated bud mutants based on factors thought to affect the duration of the apical bud growth phase. Two round bud mutants (arc18 and sac6) were found to be defective in apical actin patch localization. Several elongated bud mutants demonstrated a delay of cell cycle progression at the apical growth phase, suggesting that these mutants have a defect in the control of cell cycle progression.     

Chitin structures of the cell walls of synchronously grown virgin cells of Saccharomyces cerevisiae

The ability of a lytic β-glucanase of Arthrobacter GJM-1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin-containing fraction was employed for the isolation of chitin-rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin-rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.     

Cytoplasmic Clb2 is required for timely inactivation of the mitotic inhibitor Swe1 and normal bud morphogenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Subcellular localization is an important determinant of substrate and functional specificity for cyclin–cyclin dependent kinase (CDK) complexes. This work addresses the cytoplasmic function of the budding yeast mitotic cyclin Clb2, which is mostly nuclear but is also present in the bulk cytoplasm and at the mother-bud neck. Clb2 contains two leucine-rich nuclear export signals (NESs)—one of which we newly describe here—that maintain its presence in the cytoplasm. Yeast strains bearing mutations in one or both of these NESs have elongated buds, indicative of a G2/M cell cycle delay. A small number of these cells exhibit a filamentous-like morphology under conditions that do not normally induce filamentous growth. These phenotypes are enhanced by deletion of the other three mitotic cyclins (CLB1,3,4) and are dependent on expression of Swe1, the yeast Cdk1 inhibitory kinase. Δclb1,3,4 Δbud3 cells, which fail to localize Clb2 to the bud neck, also exhibit a Swe1-dependent elongated bud phenotype. Our results support a model in which cytoplasmic Clb2-Cdk1 is required for timely inactivation of Swe1 at the G2/M transition and bud neck targeting of Clb2 contributes to the efficiency of this process. Cytoplasmic Clb2 may also be important for repression of filamentous growth.     

Intracellular and extracellular regulation of ureteric bud morphogenesis

The urinary collecting duct system of the permanent kidney develops by growth and branching of an initially unbranched epithelial tubule, the ureteric bud. Formation of the ureteric bud as an outgrowth of the wolffian duct is induced by signalling molecules (such as GDNF) that emanate from the adjacent metanephrogenic mesenchyme. Once it has invaded the mesenchyme, growth and branching of the bud is controlled by a variety of molecules, such as the growth factors GDNF, HGF, TGFβ, activin, BMP-2, BMP-7, and matrix molecules such as heparan sulphate proteoglycans and laminins. These various influences are integrated by signal transduction systems inside ureteric bud cells, with the MAP kinase, protein kinase A and protein kinase C pathways appearing to play major roles. The mechanisms of morphogenetic change that produce branching remain largely obscure, but matrix metalloproteinases are known to be necessary for the process, and there is preliminary evidence for the involvement of the actin/myosin contractile cytoskeleton in creating branch points.     

Intracellular and extracellular regulation of ureteric bud morphogenesis

The urinary collecting duct system of the permanent kidney develops by growth and branching of an initially unbranched epithelial tubule, the ureteric bud. Formation of the ureteric bud as an outgrowth of the wolffian duct is induced by signalling molecules (such as GDNF) that emanate from the adjacent metanephrogenic mesenchyme. Once it has invaded the mesenchyme, growth and branching of the bud is controlled by a variety of molecules, such as the growth factors GDNF, HGF, TGFβ, activin, BMP‐2, BMP‐7, and matrix molecules such as heparan sulphate proteoglycans and laminins. These various influences are integrated by signal transduction systems inside ureteric bud cells, with the MAP kinase, protein kinase A and protein kinase C pathways appearing to play major roles. The mechanisms of morphogenetic change that produce branching remain largely obscure, but matrix metalloproteinases are known to be necessary for the process, and there is preliminary evidence for the involvement of the actin/myosin contractile cytoskeleton in creating branch points.     

第3～5周人胚肝的细胞特征和生长因子及受体表达的研究

用第 3～ 5周人胚 ,石蜡切片 ,免疫组化染色 ,光镜下观察人胚肝的细胞特征和HGF、IGF -I、TGFβ1等生长因子及其受体、PCNA、AFP、CK19等的表达。结果发现第 3周末肝芽形成 ,第 4周肝索开始形成 ,第 3～ 4周人胚肝由同一类具有幼稚细胞形态学特征的细胞构成。这些细胞为AFP、c Met阳性反应。第 5周时肝索细胞的数量增加 ,开始出现PCNA的表达 ,仍仅为同一类细胞。第 5周肝索细胞呈IGF -I、TGFβ1及其受体免疫反应阳性 ,HGF阴性 ,其周围的心肌细胞及间充质细胞为HGF阳性反应。结果提示第 3～ 5周 ,组成肝芽和肝索的细胞属于肝干细胞 ,其形态和因子表达的差异说明肝干细胞可能处于不同的发育阶段 ,AFP、c Met可以作为此阶段肝干细胞的标记物 ,HGF、IGF -I、TGFβ1及其受体可能参与对早期人胚肝发育的调节。     

Selective growth in culture of fetal rat renal collecting duct anlagen. Morphologic and biochemical characterization

Cell culture conditions were devised that selectively supported growth of 13 or 14 gestation day F344 rat ureteric bud, the renal collecting duct anlagen. These same conditions also inhibited the growth of metanephrogenic mesenchyme, precursor of structures proximal to the duct. Isolated buds were cultured in Ham's F12 medium supplemented with epidermal growth factor, selenium, insulin, hydrocortisone, prostaglandin E1, transferrin, and triiodothyronine; fetal bovine serum (1%) was required for continuous propagation. Cultured cells were epithelial in morphology and formed domes. By electron microscopy, many structural characteristics of highly differentiated cells were evident: numerous mitochondria, Golgi apparatus, extensive endoplasmic reticulum, an occasional cilium, intracytoplasmic filaments, polarized formation of microvilli, and gap junctions. Histochemistry revealed considerable functional differentiation as well. Cultured bud cells, adult collecting duct, and fetal duct anlagen were positive for acid phosphatase, membrane-localized ATPase, and nonspecific esterase. Bud cells and fetal duct anlagen expressed high levels of gamma-glutamyl transpeptidase activity while adult collecting duct exhibited slight activity. In addition, immunocytochemical observation of intermediate filament expression revealed the presence of epithelial cytokeratins but absence of mesenchymal vimentin in cultured bud cells and fetal and adult collecting ducts. These results indicate that the culture conditions described can maintain the partially differentiated fetal collecting duct anlagen in a state consistent with its embryonal derivation, and therefore may be useful in culture studies of renal differentiation.     

Innervation of taste buds revealed with Brainbow‐labeling in mouse

Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin‐cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non‐taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow‐labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP‐43, a marker for growing fibers and growth cones.     

Glycosaminoglycans in the basal lamina and extracellular matrix of serially aged mouse mammary ducts

The synthesis and accumulation of specific glycosaminoglycans into proteoglycans of the basal lamina and extracellular matrix is an important aspect of ductal growth and branching morphogenesis in the mouse mammary gland. The present study was undertaken to determine whether serially aged mammary gland, which has lost most of its growth potential during repeated transplantation, displays altered ability to synthesize and accumulate glycosaminoglycans into the extracellular matrix or basal lamina. Using histochemical and autoradiographic procedures coupled with enzymatic digestion, it is now shown that serially aged mammary ductal tissue synthesizes and incorporates hyaluronate into the basal lamina at the leading edge of the end bud, where growth takes place, and sulfated glycosaminoglycans are accumulated in the extracellular matrix along the end bud flanks, associated with ductal morphogenesis. These patterns of synthesis and accumulation are similar to those associated with the growth of young gland. In non-growing regions, regardless of whether growth termination resulted from serial aging or from normal growth regulatory mechanisms operating in the young gland, sulfated glycosaminoglycans were distributed in the extracellular matrix around the ductal tips. Again, the pattern was similar in young and serially aged gland. We conclude that glycosaminoglycan metablism and distribution are related to growth status rather than tissue age, and are unlikely to be an important component of mammary senescence.     

Intercalary and supernumerary regeneration in the limbs of the frog, Xenopus laevis.

Anuran amphibians, such as Xenopus laevis, can regenerate their limbs only when they are young tadpoles, whereas urodele amphibians have a regenerative ability throughout their lives. It is still unclear whether anuran and urodele use the same mechanism during regeneration. In the present study, we analyzed intercalary and supernumerary regeneration in Xenopus. In contrast to urodele blastema that induces intercalary regeneration along the proximodistal (PD) axis, intercalation did not occur in the Xenopus limb bud when the presumptive zeugopodium (fibula and tibia) was removed. However, when the limb bud tip (presumptive autopodium) was transplanted to the presumptive stylopodium (femur) with a 180-degree rotation at stage 52, the complete zeugopodium was regenerated. These results were similar to the results of urodele mature limbs, suggesting that Xenopus limb buds are equivalent to the urodele mature limbs but not to the urodele blastemas. We hypothesized that the ability for intercalation depends on the expression pattern of fibroblast growth factor (fgf)-8, because the expression of fgf-8 in the urodele spreads over the whole blastema and is close enough to activate the growth of the stump. To test this hypothesis, an FGF-8-soaked bead was implanted at the boundary between the stump and tip of a Xenopus limb bud. Intercalary regeneration was induced at stages 52 and 53. These results suggest that the Xenopus limb bud possesses the potential for intercalation, but endogenous FGF-8 in the apical ectodermal ridge (AER) does not induce intercalation to the stump because of the long distance between the AER and stump.     

Relationships between ectoderm and skeletal morphogenesis in the chick embryo limb bud

Summary When in chick embryos (H.-H. stages 22 to 25) a variously large area of ectoderm with the subjacent mesodermal layer external to the superficial vessel network, loosened from the dorsal face of the wing bud is rotated 180° in situ, or a similar ecto- and mesodermal sheet isolated from the dorsal face of the leg bud is grafted, in normal of 180° reversed orientation, onto the dorsal face of the wing bud, no changes in the normal developmental pattern of the wing skeleton ensue. As the grafted tissue, which apparently does not contain prospective chondrogenic cells, develops as a flat implant, the normal geometry of the ectodermal hull is not altered: therefore, the biomechanical conditions and the polarized growth of the skeletogenous mesenchyme of the wing bud, which seem to be controlled by the enveloping epithelium, remain practically unchanged.Morphological alterations of the skeletal pieces of the wing and formation of ectopic cartilage follow instead the implantation on the dorsal face of the wing bud, in normal or 180° reversed orientationm of an ecto- and mesodermal sheet similar to the one mentioned above but containing also a varying amount of the mesenchyme lying beneath the superficial vessel network.     

Influenza virus morphogenesis and budding

Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major sub-viral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins.Budding processes involve bud initiation, bud growth and bud release. The presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in bud initiation, bud growth and bud scission/release require involvement both viral and host components and can affect bud closing and virus release in both positive and negative ways. Among the viral components, M1, M2 and NA play important roles in bud release and M1, M2 and NA mutations all affect the morphology of buds and released viruses. Disassembly of host cortical actin microfilaments at the pinching-off site appears to facilitate bud fission and release. Bud scission is energy dependent and only a small fraction of virus buds present on the cell surface is released. Discontinuity of M1 layer underneath the lipid bilayer, absence of outer membrane spikes, absence of lipid rafts in the lipid bilayer, as well as possible presence of M2 and disassembly of cortical actin microfilaments at the pinching-off site appear to facilitate bud fission and bud release. We provide our current understanding of these important processes leading to the production of infectious influenza virus particles.     

Characteristics of initiation and early events for muscle development in the Xenopus limb bud.

In Xenopus laevis, limb buds start to develop at a later point of the larval stage, prior to metamorphosis. This onset of limb development in Xenopus is totally different from that in amniotes such as birds and mammals, in which limb buds emerge at an early stage of embryogenesis, in parallel with other organogenesis. We investigated limb myogenesis in Xenopus, focusing on myogenic gene expression, myogenic ability of limb bud cells in the early stage, and the origin of myogenic precursor cells in the limb bud. The Xenopus early limb bud contains myoD/cardiac actin-positive and pax3/pax7-negative cells. Interestingly, results of transplantation experiments have revealed that this early limb bud contains myogenic precursor cells. In order to know the contribution of myogenic cells in somites to myogenic precursor cells in the early limb bud, we used a Cre-LoxP system for tracing over a long period. The results of fate tracing for myogenic cells in somites of the Xenopus embryo suggested that early-specified myogenic cells in somites do not contribute to limb muscle in Xenopus. Taken together, the results suggest that limb muscle development in Xenopus has characteristics of initiation and early events distinct from those of other vertebrate clades.     

Both Chondroinduction and Proliferation Account for Growth of Cartilage Nodules in Mouse Limb Bud Cultures

High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules. We demonstrated that Col II-positive replicating chondrocytes are present throughout the nodules with the majority of replicating cells localized on the top (cell-medium interface) and periphery/sides of nodules. Kinetic analysis of cellular proliferation within the nodules demonstrated the time-dependent reduction in number of Col II-positive replicating cells. The sequential expression of Col I, Col II, Col X, parathyroid hormone related peptide receptor 1 (Pthr1), bone sialoprotein (Bsp) and osteocalcin (Ocn) mRNAs was similar to that characterizing chondrocyte differentiation and maturation in vivo. We conclude that the limb bud model recapitulates events seen during endochondral bone formation: cellular aggregation, proliferation, differentiation and maturation to hypertrophy. We also conclude that not only induction of peri-nodular mesenchymal cells but also proliferation of chondrocytes within cartilage nodules contribute to cartilage nodule growth.     

Control of axillary bud initiation and shoot architecture in Arabidopsis through the SUPERSHOOT gene

The aerial architecture of flowering plants is determined to a large extent by shoot growth and shoot branching arising from the initiation and growth of axillary meristems. We have identified an Arabidopsis mutant, supershoot (sps), which is characterized by a massive overproliferation of shoots, such that a single plant can generate 500 or more inflorescences. Analysis of the mutant plants shows that the primary defect is because of an increase in the number of meristems formed in leaf axils, together with release of bud arrest, resulting in reiterative branch formation from rosette and cauline leaves. The SPS gene is shown here to encode a cytochrome P450, and together with a 3- to 9-fold increase in levels of Z-type cytokinins in sps mutant plants, indicate a role for SPS in modulating hormone levels. The expression pattern of SPS, with strong expression at the leaf axils, correlates well with the phenotypic defects. Our results indicate that control of shoot branching in Arabidopsis may be accomplished in part by suppression of axillary meristem initiation and growth through the localized attenuation of cytokinin levels at sites of bud initiation.     

The 2-stage neuroskeletal pathomechanism of developmental deformities of the limb skeleton. A contribution to the discussion on the McCredie-McBride hypothesis

Experimental skeletal deformities produced in laboratory birds and in frog tadpoles and examined with Williams' technique (1943) suggest a selective inhibitory effect of various teratogens upon the vulnerable growth of peripheral nervous trunks. The exaggerated osteoneural growth differential resulting therefrom is compensated for by adaptive deformities (buckling, achondroplasic stunting, dislocation) of otherwise normally growing bones which, though independent of innervation under normal conditions, have to "respect" the growth insufficiency of the nervous trunks and to accommodate along them during the proximo-distal development of the limb, even at the cost of a gross deformity. The McCredie-McBride hypothesis, on the other hand, is aimed at explanation of skeletal defects by an early neuroskeletal (neurotrophic) disturbance within the limb bud. Aneurogenic limbs produced experimentally do not necessarily militate against the existence of neuroskeletal relations in the early limb bud postulated, above all, by the McCredie-McBride hypothesis. These relations have been firmly established during the phylogenetic history so that artificial aneurogenic limb, never evolved by Nature, may grow up by (phylo)genetic inertia even without any neural involvement during the individual ontogenesis.     

Primary culture of rat taste bud cells that retain molecular markers for taste buds and permit functional expression of foreign genes

Taste buds are constituted of several kinds of cells which have distinct characteristics and play different roles. In this study, we have established an in vitro culture system by optimizing the method for isolating the cells and by selecting culture media and reagents effective for cell viability and adhesion. As a result, the taste bud cells were adhesive and viable for over 3 days when cultured onto Matrigel-coated dishes in medium based on keratinocyte growth medium. The cells retained molecular markers for both the cytoskeleton and intracellular signaling such as cytokeratin 8 and phospholipase Cbeta2. In addition, three intracellular signaling molecules, gustducin, phospholipase Cbeta2, and inositol 1,4,5-trisphosphate receptor type 3, are expressed in the same correlation as those in vivo, although the ratio of signaling molecule-positive cells vs. total cells was somewhat lower in the culture than in vivo. Next, we tried several methods to introduce foreign genes into the cells, and obtained a greater than 90% efficiency of introduction using an adenovirus vector. Finally, we show that an exogenously expressed myc-tagged alpha1A-adrenoceptor sorts into the plasma membrane, and transduces a ligand-dependent signal resulting in intracellular [Ca(2+)] increase in about half of the infected cells.These results suggest that taste bud cells after 3 days of culture retain characteristic molecular markers, and may prove useful for describing the molecular and physiological features of taste bud cells, and that these cells can be further manipulated by adenovirus-mediated gene introduction.