Nucleolar variations during the ontogenesis of diploid and tetraploid cyprinid species

Studying the quantitative characteristics of nucleoli in cells of several cyprinid species ( Cyprinus carpio, Carassius auratus gibelio, Carassius auratus auratus, Aristichthys nobilis, Ctenopharyngodon idella, Hypophthalmichthys molitrix ) has shown a definite programme that controls genetically changes in the nucleolar characteristics during fish ontogenesis. In embryogenesis, the nucleolar activity, which has been estimated from the number and size of nucleoli, decreased; this process was realized dissimilarly in diploid and tetraploid cells. A correlation between the nucleolar characteristics and the body mass of juvenile fish was found. Parameters of the nucleolar activity were closely related to the physiological state of mature organisms and featured tissue specificity, rhythmicity, and high level of heritability.     

Tracking morphogenetic tissue deformations in the early chick embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to study the physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.     

Specification of germ layer identity in the chick gastrula

Background  

Chick definitive endoderm is an important source of signals that pattern the early embryo forming a central structure around which the body plan is constructed. Although the origin of definitive endoderm has been mapped in the chick, arising principally from rostral streak at elongating streak stages, it is not known when this layer first becomes fully committed to its germ layer fate, an important issue to resolve in light of its critical role in subsequent patterning of the early embryo.     

Expression of chick Coactosin in cells in morphogenetic movement

Coactosin is a 17 kDa actin binding protein that belongs to the actin depolymerizing factor/cofilin homology family. Coactosin inhibits barbed‐end capping of actin filament, and is involved in actin polymerization. Coactosin is expressed in cephalic and trunk neural crest cells, cranial ganglia and dorsal root ganglia. Coactosin is also expressed in the cells that are forming mesonephric duct, and endodermal cells. Immunocytochemistry with anti‐Coactosin antibody shows that Coactosin is localized in the cytoplasm, and associated with actin stress fibers in cultured neural crest cells. Coactosin is also expressed in the axon of oculomotor nerve and trigeminal nerve. In the growth cone of the oculomotor nerve axons, both Coactosin mRNA and protein were localized, which is indicative of the role of Coactosin in pathfinding of the growth cone. Coactosin is expressed in those that require dynamic and highly coordinated regulation of actin cytoskeleton, that is, neural crest cells, cells in the tip of the mesonephros, endodermal cells and axons.     

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

Dynamic morphogenetic events characterize the mouse visceral endoderm

Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.     

Telomere length in male germ cells is inversely correlated with telomerase activity

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.     

Expression of gamma-adducin is associated with regions of morphogenetic cell movement in the chick embryo

The adducin family of cytoskeletal proteins are known to mediate actin driven cell movements. Here we describe the cloning and expression pattern of a gene encoding gamma-adducin in the chick embryo. Expression of this gene is strikingly restricted to the epithelial cell layer (with a few exceptions including emerging notochord and lateral mesoderm). Gamma-adducin is detected at particularly high levels in cell populations undergoing important morphogenetic movements, such as epiblast approaching the primitive streak, regressing spinal cord primordia and closing neural tube.     

Expression of gamma-adducin is associated with regions of morphogenetic cell movement in the chick embryo

The adducin family of cytoskeletal proteins are known to mediate actin driven cell movements. Here we describe the cloning and expression pattern of a gene encoding gamma-adducin in the chick embryo. Expression of this gene is strikingly restricted to the epithelial cell layer (with a few exceptions including emerging notochord and lateral mesoderm). Gamma-adducin is detected at particularly high levels in cell populations undergoing important morphogenetic movements, such as epiblast approaching the primitive streak, regressing spinal cord primordia and closing neural tube.     

Nucleolar size and the activity of rRNA transport in the course of morphogenetic reduction of cell dimensions

In antheridial filaments of Chara vulgaris during the first period of spermatogenesis which consists of 6 synchronous cell division cycles there occurs a gradual decrease in sizes of cells entering successive mitoses. Present studies indicate that this process is correlated with a considerable reduction of total nucleolar volume in late G2 phase which, in turn, brings about decrease in sizes of nucleoli reappearing in telophase of the subsequent cell cycle. A consequence of the above phenomenon evidenced using 3H uridine autoradiography is a gradual increase--from one generation to the next--in an amount of rRNA transported into cytoplasm due to an increase in number of small nucleoli which were found to be more active in transport than larger nucleoli. This process leads to a lowering of an increase in nucleolar volumes during consecutive interphase periods owing to a progressively limited accumulation of rRNA for the sake of daughter cells, i.e. to a spontaneously magnifying reduction of nucleolar sizes in the forthcoming cell generations. Thus, diminution of nucleoli observed during the development of antheridial filaments seems to be due to a chain-series of processes connected with mechanisms which possibly regulate rRNA transport and accompany morphogenetic events.     

Development of germ cells in chick ovarian medulla]

Ovaries from chick embryos and chicken have been investigated with a view to the evolution of germ cells in the medullary. They can enter meiosis and reach pachytene. They seem to be eliminated by the way of the lacunas. Some observations can be utilized in a discussion about the initiation of the meiosis and the evolution of the ovocytes.