QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Movies Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carré, D. Articles by Sardet, C. Search for Related Content PubMed PubMed Citation Articles by Carré, D. Articles by Sardet, C.

Formation of a large Vasa-positive germ granule and its inheritance by germ cells in the enigmatic Chaetognaths

Bio Mar Cell, Laboratoire de Biologie du Développement, UMR7009 CNRS / UPMC, Station Zoologique, Observatoire Océanologique, 06234 Villefranche-sur-mer cedex, France

View larger version (133K): [in a new window]   Fig. 1. Fertilization in chaetognaths. (A) Spadella cephaloptera: adult specimen with the two testes (T) and the two ovaries (O) in the posterior and anterior part of the body cavity, respectively. The two rows of maturing oocytes are colored pink and the two seminal receptacles (SR) and sperm ducts (SD) are colored blue. (B) Sagitta inflata: oocyte (pink) just before fertilization. The germinal vesicle (GV) has migrated to the animal pole opposite the fertilization apparatus (yellow). (C) Sagitta inflata: a portion of the sperm duct (SD) filled with sperm (blue). Bundles of sperm (arrows) traverse the sperm duct wall in the direction of oocytes. (D) Sagitta inflata: first polar body (PB) emission just before fertilization. (E) Sagitta inflata: a bundle of sperm (blue) reaches the fertilization apparatus. The AFC2 cell is colored yellow. The nucleated portion of AFC2 cell contacts the oocyte (pink) while the rest of the cell spirals (arrow) and contacts the sperm bundle (blue). (F) Sagitta setosa: bundles of sperm (Sp, blue) traverse the sperm duct (SD) wall in the direction of the microtubule (Mt)-filled cellular extension of the AFC2 cell rich in vesicles (arrow). (G,H) Sagitta setosa: close-up view of the cellular extension of the AFC2 cell showing the microtubule bundles before fertilization (G) and the abundance of vesicles just after fertilization (H). (I) Sagitta bipunctata: sequence of sperm penetration in the region of the AFC2 cell (only the nuclear portion is colored yellow). The AFC2 cell resorbs. (J) Sagitta bipunctata: same as I but at a higher magnification. The entering sperm (Sp) can be observed.

View larger version (189K): [in a new window]   Fig. 2. Assembly and composition of the germ granule (see sequences on our web site at: http://www.obs-vlfr.fr/biomarcell.html, then click on Chaetognaths). (A) Sagitta setosa: During the migration of the male pronucleus (MP) and female pronucleus (FP) 25 minutes after fertilization aggregation of cortical particles (arrow) begins in the vegetal cortex and leads to the formation of the germ granule (GG). During cleavage the germ granule (GG) is segregated into one of the two first blastomeres. PB, polar body. (B) Sagitta bipunctata: sequence of images, spaced about 2 minutes apart, showing the aggregation of the small cortical particles (arrows) into a single germ granule. (C,D) Sagitta bipunctata: vegetal cortex just after egg laying (C) and during pronuclear migration (D). Electron-dense particles are first dispersed along the vegetal cortex (arrows in C) and gather together (D). (E) Sagitta bipunctata: thin section of the germ granule situated near the vegetal cortex at the 2-cell stage (PM, plasma membrane). The germ granule is a compact and well-delineated aggregate of electron-dense material derived from the cortical particles and contains endoplasmic reticulum (ER) and mitochondria (M). (F) Sagitta setosa: germ granule in vivo seen with DIC optics: a granular substructure can be observed. (G,H) Spadella cephaloptera: immunolocalization (peroxidase stained) of Vasa protein in the germ granule at the 2-cell stage. The substructure can be seen in H (Drosophila anti-Vasa, rabbit polyclonal antibody). (I) Spadella cephaloptera: immunoblot of Drosophila embryos (left) and Spadella embryos (right) showing a cross-reaction with the Drosophila anti-Vasa antibody. A major band of approx. 70 kDa can be seen in Drosophila and in Spadella samples.

View larger version (128K): [in a new window]   Fig. 3. Formation of germ granule in fertilized bisected egg fragments of Sagitta bipunctata. (A) Equal section along the animal-vegetal (AV) axis: the fragment containing the two pronuclei (left) has segmented into four closely apposed cells whose boundaries are not seen in the plane of focus shown (two of the four nuclei are indicated by arrows). The germ granule (GG) has formed in the vegetal cortex of both nucleated (left) and enucleated (right) fragments. (B) Unequal section along the AV axis: germ granules (GG) have sizes proportional to the size of vegetal hemisphere fragments (see inset). (C,D) Same two fragments in two different planes of focus after performing a slightly oblique section with respect to the AV axis (see inset). In C it can be seen that each fragment contains a pronucleus (MP, FP). In the tangential focal plane shown in D, two germ granules (GG) are seen forming in the cortex.

View larger version (145K): [in a new window]   Fig. 4. Relocalisation of the germ granule during segmentation in Sagitta inflata. The germ line blastomere and the germ granule are coloured in two shades of green. (A,B) The germ granule (arrowhead) is cortical and juxtaposed to the cleavage plane during stages 2 (A) and 4 (B). (C-E) The sequence C to D shows the migration of the germ granule (arrowhead) towards the nucleus (N) during stage 4-8. During telophase in E, the germ granule is seen stretching towards one of the spindle poles. It is inherited by one of the two blastomeres at stage 8. (F) Stage 8. As in E, the germ granule (arrowhead) is lying on the side of the spindle and is closer to one of the spindle poles (compare to E). (G,H) Stage 16. The germ granule (arrowhead) forms a cap around one spindle pole (G; live observation in DIC) (H; thick section tangential to vegetal pole). (I) Stage-31 cells. All blastomeres have divided except for the larger cell containing the germ granule (light green). (J) Stage-32 cells. Unequal cleavage segregates the germ granule (arrowhead) into the smallest blastomere constituting the founder PGC. (K) Stage-32 cells. The germ granule cannot be seen in vivo but the founder PGC is identifiable because it remains in interphase with a large visible nucleus. (L) Stage-63 cells. All blastomeres divide except for the founder PGC. (M) Stage 64 cells. The founder PCG has divided into two cells identifiable by the continuous presence of an interphase nucleus. (N) Blastula stage.

View larger version (185K): [in a new window]   Fig. 5. PGCs during embryogenesis. (A) Spadella cephaloptera: sequence of four images showing the beginning of gastrulation. The first two PGCs are identified by the continuous presence of large interphase nuclei (colored ochre). During gastrulation these cells are the first to invaginate with the archenteron (arrows in second image) and to position themselves at the tip of the archenteron (last image of the sequence). (B,C) Sagitta inflata: late gastrula. The two first PGCs have divided once. They are identified by their well-delineated interphase nuclei. (D) Sagitta bipunctata: as the trunk of the embryo elongates, PGCs migrate (three PGCs are visible due to their large interphase nuclei). (E) Sagitta bipunctata: thin section through one of the PGC at gastrula stage (see sequence A). Many electron-dense islands (arrow) are dispersed in the cytoplasm. N, nucleus of the PGC. (F) Sagitta bipunctata: median portion of a juvenile showing the four PGCs positioned in pairs above and below the transversal septum (TS).

View larger version (205K): [in a new window]   Fig. 6. Gametogenesis. (A) Sagitta bipunctata: two PGCs above the transversal septum (TS) will give the female germ cells of the two ovaries; the two PGCs below will yield the male germ cells of the two testes (see also Fig. 5F). (B) Sagitta bipunctata: the four PGCs (arrows) are labelled with Drosophila anti-Vasa antibody. (C) Sagitta bipunctata: the four PGCs have proliferated (4 days after fertilization). (D) Sagitta bipunctata: thin section of one of the four first PGCs. Characteristic electron-dense material (arrows) is sandwiched between the nucleus (N) and a ring of mitochondria (M). (E) Spadella cephaloptera: thin section of proliferating PGCs containing dispersed electron-dense material (arrows). (F) Spadella cephaloptera: Drosophila anti-Vasa antibody localization in oocytes (peroxidase detection). (a,b) The GVs of oocytes are surrounded by patches of Vasa-positive material. Three of these patches are indicated by arrows in b. (c) A higher magnification view of the Vasa-positive patches (arrows) at the level of the GV surface (tangential view). (d) Control; view of a GV stage oocyte exposed to HRP-labelled secondary antibody only. (G) Sagitta inflata: thin section of a portion of an oocyte GV showing the electron-dense material on both side of the nuclear pore-rich GV membrane (arrows). (H) Spadella cephaloptera: Immunoblots with Drosophila anti-Vasa polyclonal antibody using three eggs, three embryos or three juveniles, representing similar amounts of protein in each lane. An increasing amount of Vasa-like protein is visualized using two different detection methods (ECL/Vectastain peroxidase kit).

View larger version (42K): [in a new window]   Fig. 7. Germ plasm and germ cells during the life cycle of chaetognaths. During oogenesis (1), germ plasm/nuage material (in green) is within and around the germinal vesicle (GV). During maturation and internal fertilization at the vegetal pole (2), germ plasm presumably fragments into minute granules. After spawning (3), many small granules line the vegetal cortex (V) and then aggregate during amphimixy (4). At mitosis (5), small germ granules aggregate into a single large granule. This large granule is segregated into one of the first two blastomeres and continues to be inherited by only one vegetal blastomere until the 32-cell stage (7). The germ granule then fragments and is distributed into two blastomeres at the 64-cell stage (8). The germ plasm is then found in the four presumptive PGCs at the tip of the archenteron in the gastrula (9). The four PGCs become the male (posterior) and female (anterior) germ cells in the juvenile (10), which give rise to the spermatocytes and the oocytes in the adult.