Syncytial Cytoplasm Research Articles

Serous Cutaneous Glands in Phyllobates bicolor (Anura: Dendrobatidae): An Ontogenetic, Ultrastructural Study on Secretory Product Biosynthesis and Maturation

Secretory activity of serous (poison) cutaneous glands was studied with light and transmission electron microscopy over the ontogenetic cycle of the Black-legged Dart Frog Phyllobates bicolor. Serous gland activity includes two steps: biosynthesis and maturation. Biosynthesis is performed by the rough endoplasmic reticulum, smooth endoplasmic reticulum, and Golgi apparatus, and follows the usual pathways of exocrine secretory processes. Maturation is typical of anuran serous glands, which accumulate their products in syncytial secretory units. Maturation involves merging processes between secretory deposits (vesicles and/or granules) distally from the Golgi stacks, as well as an increase in the surface area between the syncytial cytoplasm and the secretory vesicles/granules. In P. bicolor, this interface area increases through the formation of peculiar cytoplasmic outgrowths. These outgrowths allow transport processes involved in maturational changes of secretory product. During maturational storage, the secretory products of P. bicolor acquire a repeating substructure of spherical to short tubular subunits. This recurrent pattern is seen in cutaneous poisons of several anuran species, regardless of their phylogenetic relationships. Among anurans, condensing serous cutaneous products into repeating aggregations of subunits appears to be a successful strategy, which allows intracytoplasmic accumulation of pharmacologically active and/or toxic molecules through various post-Golgian processes. The present findings provide evidence of a novel repertory of cytological activities that stress the large diversity of the maturational pathways leading to the peculiar repeating arrangement of secretory subunits.

Ultrastructural studies on egg envelopes surrounding the miracidia of Mediogonimus jourdanei Mas-Coma et Rocamora, 1978 (Digenea, Microphalloidea, Prosthogonimidae)

AbstractThe intrauterine, mature and fully embryonated eggs of the prosthogonimid trematode Mediogonimus jourdanei Mas-Coma et Rocamora, 1978 were examined by means of transmission electron microscopy (TEM), using high pressure freezing, freeze substitution and infiltration with resin techniques. Each embryonated egg is composed of a miracidium surrounded by three envelopes: (1) the egg shell, (2) the outer and (3) inner envelopes. Egg envelopes play an important role in the protection, metabolism, storage of nutritive reserves and the general biology of the M. jourdanei egg. The inner envelope is characterised by large, flattened nuclei, and its syncytial cytoplasm contains a heavy accumulation of glycogen, lipid droplets, mitochondria and large vesicles. These traits indicate that this layer has the features of a metabolically-active syncytial layer with an energy storage capability. In the infective eggs observed before the hatching of the miracidium, areas of so-called “focal cytoplasmic degradation” were frequently observed, which may be involved in the autolytic process of all components of this envelopes.

Separation of the Syncytial Layer of Spargana using Urea

The tegument of tapeworms is known to be composed of an outer syncytial cytoplasm layer which includes microtriches and cytoplasmic organelles (= syncytial layer), and a parenchymatous cytoplasm layer that contains subtegumental cell nuclei (= subtegumental layer) and organelles. In the present study, separation of the syncytial layer of the sparganum, the plerocercoid stage of Spirometra mansoni, was tried using urea as the chemical reagent. Histological sections were prepared to visualize the status of separation after staining with hematoxylin and eosin. The results showed that the syncytial layer of the sparganum tegument which includes microtriches and cytoplasmic organelles were successfully separated from the parenchyma using 3 M urea.

Chemical skin defence in the Eastern fire-bellied toad Bombina orientalis: an ultrastructural approach to the mechanism of poison gland rehabilitation after discharge

Type I serous glands in the skin of the Eastern yellow-bellied toad Bombina orientalis released their product massively after 10 -3 M nor-adrenalin (NA) stimulation, mimicking orthosympathetic control on poison emission in chemical skin defence. Features of cutaneous glands involved in this bulk discharge were observed under light and electron microscopes. Furthermore, restoration of depleted glands was followed after 1, 2 and 3 weeks, and compared with serous biosynthesis during larval gland development. Bulk discharge was caused by contraction of myoepithelial cells (mecs) encircling the secretory units. Mec compression dramatically affected the secretory unit, but parts of this syncytial cytoplasm were saved from degeneration and cooperated in gland renewal with stem cells from the gland neck. These adenoblasts underwent proliferation and secretory cytodifferentiation, until merging with the syncytium. Cytoplasm that had resumed secretory activity showed the features typical of larval gland development: the endoplasmic reticulum (rer) cisterns were aligned in close parallel arrangement and Golgi stacks released minute type I granules. Secretory rehabilitation led to increasing amounts of granule content. In the meantime, rough cisterns decreased in number and assumed the less ordered pattern described in control specimens. Data collected in the present study revealed that chemical skin defence in anurans is a multi-factorial mechanism involving specific activities: mechanical from mecs, biosynthetic from secretory syncytium and proliferative from intercalated stem cells.

Placental protein 13 (galectin-13) has decreased placental expression but increased shedding and maternal serum concentrations in patients presenting with preterm pre-eclampsia and HELLP syndrome

Placental protein 13 (PP13) is a galectin expressed by the syncytiotrophoblast. Women who subsequently develop preterm pre-eclampsia have low first trimester maternal serum PP13 concentrations. This study revealed that third trimester maternal serum PP13 concentration increased with gestational age in normal pregnancies (p < 0.0001), and it was significantly higher in women presenting with preterm pre-eclampsia (p = 0.02) and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome (p = 0.01) than in preterm controls. Conversely, placental PP13 mRNA (p = 0.03) and protein, as well as cytoplasmic PP13 staining of the syncytiotrophoblast (p < 0.05) was decreased in these pathological pregnancies compared to controls. No differences in placental expression and serum concentrations of PP13 were found at term between patients with pre-eclampsia and control women. In contrast, the immunoreactivity of the syncytiotrophoblast microvillous membrane was stronger in both term and preterm pre-eclampsia and HELLP syndrome than in controls. Moreover, large syncytial cytoplasm protrusions, membrane blebs and shed microparticles strongly stained for PP13 in pre-eclampsia and HELLP syndrome. In conclusion, parallel to its decreased placental expression, an augmented membrane shedding of PP13 contributes to the increased third trimester maternal serum PP13 concentrations in women with preterm pre-eclampsia and HELLP syndrome.

Cubic crystal protein inclusions in the neodermis of the pancreatic fluke, Eurytrema pancreaticum, and Eurytrema coelomaticum

Light microscopy of Eurytrema pancreaticum and Eurytrema coelomaticum collected from cattle in Japan, China, Thailand, and Brazil showed many cubic crystal inclusions in the neodermis (tegument) of all flukes. The crystal inclusions were histochemically positive for protein. Scanning electron microscopy showed many cubic protrusions containing cubic crystal protein inclusions on the surface of the neodermis. Transmission electron microscopy showed that cubic crystal protein inclusions appeared in the perikarya of subtegumental parts, passed through the cytoplasmic bridge, moved into the syncytial neodermal cytoplasm, and then protruded from, and finally separated from, the neodermal cytoplasm. Cubic crystal protein inclusions were hexahedral with each side 2-18 microm long. High-resolution microscopy of ultrathin sections of crystal inclusions showed a lattice fringe at spacings of about 0.52 nm by using a filtering processing. Diffractograms were obtained by Fourier transform of the images. The lattice structure of the crystal protein inclusions was shown by inverse Fourier transform, indicating that the cubic crystal protein inclusions were single crystals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis estimated the molecular weight of protein in the cubic crystal inclusion as 36.6 kDa. Energy-dispersive X-ray spectroscopy proved that the cubic crystal protein inclusions were composed of protein and sulfur.

Ultrastructure of the ovary, ovicapt and oviduct of the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890)

AbstractThe ultrastructural details are presented of the ovary, ovicapt and oviduct of the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890) from the intestine of the sand sole Solea lascaris. Oogonia, maturing oocytes and mature oocytes are surrounded by a syncytial interstitial cytoplasm, one of the distinctive traits of which is the presence of numerous myelin-like bodies. Oocyte inclusions comprise cortical granules and a small number of lipid droplets. The thickened, nucleated epithelium of the ovicapt lacks any apical structure and is a prolongation of the narrow ovarian epithelium. The muscular sphincter of the ovicapt is formed by a band of longitudinal muscles and bands of radial muscles at right angles to the longitudinal layer, and numerous myocytes surround the ovicapt wall. The oviduct of D. rudolphii is subdivided into three regions: (1) the proximal oviduct; (2) the fertilization chamber — the region distal to the point of entry of the duct from the seminal receptacle; and (3) the ovovitelline duct — the region distal to the point of entry of the duct from the vitelline reservoir. A comparative analysis is made between the structures of the ovary, ovicapt and oviduct of D. rudolphii and those of two other spathebothriideans, Cyathocephalus truncatus and Diplocotyle olrikii, with a discussion of ultrastructural traits that might be used as taxonomic criteria within the order Spathebothriidea.

The subplacenta of the red-rumped agouti (Dasyprocta leporina L)

BackgroundHystricognath rodents have a lobed placenta, comprising labyrinthine exchange areas and interlobular trophoblast. These correspond to the labyrinthine and spongy zones of other rodent placentae. Beneath them, however, is a structure unique to hystricognath rodents called the subplacenta. We here describe the subplacenta of the red-rumped agouti and examine the possible functional correlates of this structure.MethodsPlacentae were collected from early in midgestation to near term of pregnancy and examined by standard histological techniques, immunohistochemistry and transmission electron microscopy. In addition, to study the microvasculature of the subplacenta, vessel casts were inspected by scanning electron microscopyResultsIn the subplacenta, lamellae of connective tissue support a layer of mononuclear cytotrophoblast cells. Beneath this is found syncytiotrophoblast. Clusters of multinuclear giant cells occur in the transition zone between the subplacenta and decidua. There are prominent intercellular spaces between the cytotrophoblast cells. The basal membrane of these cells is often close to fetal blood vessels. The syncytiotrophoblast surrounds an extensive system of lacunae. Microvilli project into these lacunae from the plasma membrane of the syncytiotrophoblast. The syncytial cytoplasm contains electron-dense granules. This is probably the amylase-resistant PAS-positive material identified by histochemistry. The subplacenta is supplied entirely from the fetal circulation. Within it the vessels pursue a tortuous course with sinusoidal dilatations and constrictions.ConclusionThe functions that have been attributed to the subplacenta include hormone production. Our findings are consistent with this interpretation, but suggest that hormone secretion is directed towards the fetal circulation rather than the maternal tissues.

Microsporidia Infect the Liophloeus lentus (Insecta, Colepotera) Ovarioles, Developing Oocytes and Eggs

In the ovarioles of Liophloeus lentus (Insecta, Coleoptera, Curculionidae) two types of bacteria and parasitic microorganisms belonging to Microsporidia have been found. This study shows that the different microsporidian life stages (meronts, sporonts, sporoblasts and spores) infect the outer ovariole sheath, trophic chambers, follicular cells, late previtellogenic and vitellogenic oocytes and eggs. In trophic chambers the parasites are very abundant and are distributed unevenly, i.e. their large mass occupies the syncytial cytoplasm between the nurse cell nuclei, whereas the neck region of the trophic chamber (which houses young oocytes, prefollicular cells and trophic cords) is almost free of parasites. The developing oocytes and eggs contain a lower number of parasites which are usually distributed in the cortical ooplasm. The gross morphology of the ovaries is similar in infected and non-infected specimens. Similarly, the presence of a parasite seems to not disturb the course of oogensis. The only difference was found in the ultrastructure of mitochondria in young previtellogenic oocytes. In the infected females they are unusual i.e. bigger and spherical with tubullar cristae, whereas in the non-infected insects they are elongated and have lamellar cristae. As oogenesis progresses the unusual mitochondria rapidly change their morphology and become similar to the mitochondria in non-infected females. Taking into account the distribution of parasites within the ovarioles, it is suggested that they infect growing oocytes via outer ovariole sheath and follicular epithelium rather than via trophic cords.

Pituicytoma incidentally found at autopsy

Pituicytoma is a rare benign neoplasm, occurring in the sellar and suprasellar regions. Reported herein is a case of asymptomatic pituicytoma, discovered at autopsy, in a 54-year-old Japanese woman. This is the first case report of pituicytoma, found incidentally at autopsy (incidentaloma), in which whole-mounted sections are available for histological and immunohistochemical studies. Grossly, the bisected pituitary gland revealed a round, white to light tan, 7 mm-diameter nodule. Microscopically, whole-mounted sections revealed a well-circumscribed nodule with no fibrous capsule, located mainly in the neurohypophysis and partially compressing the adenohypophysis. The tumor was composed primarily of bipolar, occasionally unipolar, cells with syncytial fibrillary cytoplasm, arranged in short curvilinear fascicles and/or storiform patterns. Unusual histological features were seen, which included a few groups of large pleomorphic tumor cells with abundant, glassy, eosinophilic cytoplasm, occasionally associated with multinucleated giant tumor cells, and scattered Herring bodies within the tumor. Immunohistochemically, the tumor showed diffuse strong expression of glial fibrillary acidic protein, S-100 protein, and vimentin. Epithelial membrane antigen immunoreactivity was focally observed, mainly in the large tumor cells. Distinction from other intrasellar tumors (granular cell tumor and pilocytic astrocytoma) is important. Because the immunohistochemical profiles of these tumors are similar, histological findings are crucial for distinction.

Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function

The mammalian neprilysin (NEP) family members are typically type II membrane endopeptidases responsible for the activation/inactivation of neuropeptides and peptide hormones. Differences in substrate specificity and subcellular localization of the seven mammalian NEPs contribute to their functional diversity. The sequencing of the Drosophila melanogaster genome has revealed a large expansion of this gene family, resulting in over 20 fly NEP-like genes, suggesting even greater diversity in structure and function than seen in mammals. We now report that one of these genes (Nep2) codes for a secreted endopeptidase with a highly restricted pattern of expression. D. melanogaster NEP2 is expressed in the specialized stellate cells of the renal tubules and in the cyst cells that surround the elongating spermatid bundles in adult testis, suggesting roles for the peptidase in renal function and in spermatogenesis. D. melanogaster NEP2 was found in vesicle-like structures in the syncytial cytoplasm of the spermatid bundles, suggesting that the protein was acquired by endocytosis of protein secreted from the cyst cells. Expression of NEP2 cDNA in D. melanogaster S2 cells confirmed that the peptidase is secreted and is only weakly inhibited by thiorphan, a potent inhibitor of human NEP. D. melanogaster NEP2 also differs from human NEP in the manner in which the peptidase cleaves the tachykinin, GPSGFYGVR-amide. Molecular modelling suggests that there are important structural differences between D. melanogaster NEP2 and human NEP in the S1' and S2' ligand-binding subsites, which might explain the observed differences in inhibitor and substrate specificities. A soluble isoform of a mouse NEP-like peptidase is strongly expressed in spermatids, suggesting an evolutionarily conserved role for a soluble endopeptidase in spermatogenesis.

An Image Analysis Technique for the Investigation of Variations in Placental Morphology in Pregnancies Complicated by Preeclampsia With and Without Interauterine Growth Restriction

The purpose of this study was to use visual image analysis to observe changes in the morphology and composition of placental villi in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). Placental biopsies from nine normal pregnancies, five cases of PE, five cases of IUGR, and five cases of PE with IUGR (PE x IUGR) were randomly sampled. Formalin-fixed, wax-embedded sections were stained with hematoxylin and eosin (H&E) and subjected to image analysis. The placental areas occupied by villi, syncytiotrophoblast, and syncytial cytoplasm and nuclei were quantified. Significantly smaller placentas were obtained from growth-restricted pregnancies. PE, with and without IUGR, had no effect on the total area occupied by villi or intervillous space. IUGR alone showed a real and consistent reduction in villous area (56.0 +/- 2.4% vs 43.6 +/- 3.3%, P <.03). While the ratio of syncytial to villous areas were noticeably reduced in all cases of PE (0.38 +/- 0.03 vs 0.24 +/- 0.07, P <.05), this ratio remained unchanging in IUGR. Birth weight was positively correlated to both placental size and total villous area occupied. Moreover, increasingly positive relationships were recorded between both syncytiotrophoblast area and syncytiotrophoblast cytoplasm and birth weight (P <.01 and P <.001, respectively). These measurements point to impoverished villus development in idiopathic IUGR. The observed changes in PE with IUGR were more akin to PE without growth restriction than IUGR alone. This suggests that idiopathic IUGR and IUGR in PE have a separate etiology, idiopathic IUGR arising through a reduction in villous area alone, and IUGR in PE caused by changes in syncytiotrophoblast quantity, more specifically the amount of syncytiotrophoblast cytoplasm.

Cutaneous myoepithelioma: a clinicopathologic and immunohistochemical study of 14 cases

Analogous to mixed tumors of salivary glands (“ pleomorphic adenomas” ), cutaneous mixed tumors (“ chondroid syringomas” ) contain a ductal (epithelial) component and a variably prominent myoepithelial component. Tumors showing purely myoepithelial differentiation (myoepitheliomas) have only recently been recognized to arise in the dermis, and to date very few cases have been described. To characterize these tumors further, 14 cutaneous myoepithelial tumors were retrieved from the authors’ consult files. Eleven patients were male and 3 were female; their median age was 22.5 years (range, 10 to 63 years), and 7 patients were between 10 and 20 years old. Tumor size ranged from 0.5 to 2.5 cm (mean, 1.1 cm). Most tumors arose on the extremities: 6 on the upper limbs, 6 on the lower limbs, and 1 each on the back and nose. Ten tumors were limited to the dermis, and 5 also extended into superficial subcutis. Thirteen tumors were myoepitheliomas (lacking ductal differentiation), and 1 tumor was a myoepithelial carcinoma (exhibiting severe cytological atypia and a high mitotic rate). Histologically, 7 tumors were solid, composed of ovoid to spindled, histiocytoid, or epithelioid cells with no significant stroma, and 7 were predominantly lobulated, with cords or nests of epithelioid, plasmacytoid, or spindled cells with a variably reticular architecture and a chondromyxoid or collagenous/hyalinized stroma. One tumor was composed solely of plasmacytoid (hyaline) cells, and 1 exhibited extensive adipocytic differentiation. Among the 13 myoepitheliomas, mitoses ranged from 0 to 6 per 10 high-power fields (HPFs) (mean, 1.5); 8 tumors contained no mitoses. The myoepithelial carcinoma had 39 mitoses per 10 HPFs. By immunohistochemistry, all cases were reactive for epithelial markers (keratins and/or epithelial membrane antigen [EMA]); 13 of 14 (93%) expressed S-100 protein, 10 of 11 expressed (91%) calponin, 11 of 14 (79%) expressed EMA, 9 of 14 (64%) expressed keratins, 8 of 14 (57%) expressed smooth muscle actin, 7 of 14 (50%) expressed glial fibrillary acidic protein, 3 of 11 (27%) expressed p63, and 1 of 6 (17%) expressed desmin. All 5 cases without keratin staining were diffusely positive for EMA, and all of these cases showed a solid growth pattern. Follow-up was available for 8 patients (median follow-up, 40 months; range, 6 months to 9 years); 3 tumors (38%) recurred locally, and 1 tumor (13%) also metastasized to the lymph nodes. The case that resulted in recurrence and metastasis had the highest mitotic rate (6 per 10 HPFs) of the cytologically benign tumors. Follow-up information was not available for the myoepithelial carcinoma. This study suggests that approximately 50% of cutaneous myoepitheliomas are distinctive lesions composed of a solid proliferation of cells with abundant eosinophilic syncytial cytoplasm, which often lack immunostaining for keratin, whereas the remainder demonstrate focally reticular architecture and myxoid stroma or plasmacytoid cells, similar to their counterparts in salivary gland and soft tissue. Whereas most cutaneous myoepitheliomas behave in a benign fashion, there is apparently a significant risk for local recurrence but a low metastatic potential.

Ultrastructure of poison glands of South American frogs: A comparison betweenPhysalaemus albonotatus andLeptodactylus chaquensis (Anura: Leptodactylidae)

Serous (poison) cutaneous glands of the leptodactylid species Physalaemus albonotatus and Leptodactylus chaquensis were compared using light and transmission electron microscopy. Glands in the two species share structural traits common in anurans, including the peripheral contractile sheath (myoepithelium) and the syncytial secretory unit that produces, stores, and modifies the poison. At the ultrastructural level, early steps of poison production are also similar and fit the usual path of proteosynthesis, involving rough endoplasmic reticulum (RER) and Golgi stacks (dictyosomes) in the peripheral syncytial cytoplasm. However, several differences are obvious during the maturational processes that lead post-Golgian products to their ultimate ultrastructural traits. In P. albonotatus, the dense product released from the dictyosomes acquires a thick repeating substructure, which, however, becomes looser in the inner portion of the syncytium. In L. chaquensis, serous maturation involves gradual condensation, and opaque, somewhat "vacuolized" granules are formed. These different maturational paths expressed during poison manufacturing in the two species agree with the polyphyletic origin of the family Leptodactylidae. On the other hand, data collected for P. albonotatus fit previous findings from P. biligonigerus and stress the view that poisons produced by congeneric species share similar (or identical) ultrastructural features.

Immuno-electron Microscopic Localization of Endothelial Nitric Oxide Synthase in Human Placental Terminal Villous Trophoblasts—Normal and Pre-eclamptic Pregnancy

We demonstrated the subcellular localization of endothelial nitric oxide synthase (eNOS) in human placental terminal villous trophoblasts at near term period, and compared the distribution pattern with that in pre-eclamptic trophoblasts, using immunogold electron microscopy. Immunolabelling for eNOS was visible markedly in the syncytial microvilli and syncytial cytoplasm. Semiquantitative analysis showed that the concentration and the distribution pattern of gold particles for eNOS did not significantly differ between normal and pre-eclamptic placental trophoblasts. These results indicated that syncytiotrophoblastic microvilli and cytoplasm were the subcellular localization sites of syncytium-derived eNOS in terminal villi, and that there were no significant differences in this eNOS subcellular distribution pattern between normal and pre-eclamptic syncytiotrophoblasts in regard to immunohistochemically detectable eNOS.

Secretory granule-cytoplasm relationships in serous glands of anurans: ultrastructural evidence and possible functional role

A survey covering the serous (granular) cutaneous glands in several anuran families from the Old and New Worlds (Bombinatoridae, Discoglossidae, Ranidae, Hylidae, Pseudidae and Leptodactylidae) has revealed consistent patterns of complex interactions between the syncytial secretory unit and serous deposits (granules). These relationships involve outgrowths from the syncytial cytoplasm encircling the granules and complex invaginations of the perigranular compartment (halo) into the syncytium. The outgrowths are branched, cytoplasm processes resembling ramified microvilli, or can be larger, dome-like to cylindrical structures. Despite their different features and origins, all these structures are efficient devices for amplifying the cytoplasmic surfaces round the granules, so improving exchange between the secretory syncytium and serous product. These complex secretory granule–cytoplasm interactions affect the product released from the Golgi apparatus and are consistent with the hypothesis of prolonged serous maturation following the initial phase of biosynthesis. Post-Golgian maturation modifies the secretory material on a centripetal gradient, causing condensation and, possibly, the transfer of component molecules from and/or to the cytoplasm.

Granular cutaneous glands in the frog Physalaemus biligonigerus (Anura, Leptodactylidae): comparison between ordinary serous and ‘inguinal’ glands

Beside the ordinary granular (or serous) glands, the skin of the leptodactylid frog Physalaemus biligonigerus possesses peculiar clusters of large granular units, the ‘inguinal’ glands, located in the dorsolateral areas of the pelvic girdle. Both gland types store their specific products within the syncytial cytoplasm of the secretory unit. These secretory materials consist of spheroidal or ellipsoidal bodies (granules) with a repeating substructure. The subcellular features of the immature products of the ordinary serous and inguinal glands are identical. However, these products undergo divergent maturative processes, leading to fluidation on the one hand and condensation on the other. Secretory release into the small gland lumen was observed in both cases, involving merocrine mechanisms. On the basis of the analysis of cutaneous serous gland polymorphism in anurans, the inguinal units in P. biligonigerus do not appear to be an independent line. Rather, these large units belong to the ordinary serous type and represent a gland population specialized in the storage of remarkable amounts of product used in chemical defence of the skin.

Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila.

Cytoplasmic dynein is a multisubunit minus-end-directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immunofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.

Development of endosperm in Arabidopsis thaliana

The process of endosperm development in Arabidopsis was studied using immunohistochemistry of tubulin/microtubules coupled with light and confocal laser scanning microscopy. Arabidopsis undergoes the nuclear type of development in which the primary endosperm nucleus resulting from double fertilization divides repeatedly without cytokinesis resulting in a syncytium lining the central cell. Development occurs as waves originating in the micropylar chamber and moving through the central chamber toward the chalazal tip. Prior to cellularization, the syncytium is organized into nuclear cytoplasmic domains (NCDs) defined by nuclear-based radial systems of microtubules. The NCDs become polarized in axes perpendicular to the central cell wall, and anticlinal walls deposited among adjacent NCDs compartmentalize the syncytium into open-ended alveoli overtopped by a crown of syncytial cytoplasm. Continued centripetal growth of the anticlinal walls is guided by adventitious phragmoplasts that form at interfaces of microtubules emanating from adjacent interphase nuclei. Polarity of the elongating alveoli is reflected in a subsequent wave of periclinal divisions that cuts off a peripheral layer of cells and displaces the alveoli centripetally into the central vacuole. This pattern of development via alveolation appears to be highly conserved; it is characteristic of nuclear endosperm development in angiosperms and is similar to ancient patterns of gametophyte development in gymnosperms.

An anteroposterior Dorsal gradient in the Drosophila embryo.

Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. We investigate the consequences of expressing a constitutively activated form of the Toll receptor, Toll(10b), in anterior regions of the early embryo using the bicoid 3' UTR. Localized Toll(10b) products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo. This study also provides evidence that neurogenic repressors may be important for the establishment of the sharp mesoderm/neuroectoderm boundary in the early embryo.