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

First published online 16 February 2005
doi: 10.1242/dev.01701

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development 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 Google Scholar Google Scholar Articles by Hejnol, A. Articles by Schnabel, R. Search for Related Content PubMed PubMed Citation Articles by Hejnol, A. Articles by Schnabel, R.

The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations

Technische Universität Braunschweig, Institut für Genetik, Spielmannstrasse 7, D-38106 Braunschweig, Germany

View larger version (91K): [in a new window]   Fig. 2. Cleavage up to the blastula stage in Thulinia stephaniae. The columns from left to right show embryos 1, 2 and 3 respectively. (A-C) Cell lineages from the two-cell to the 122-cell stage as viewed in SIMI°BioCell. Red dots indicate the positions where cells were marked, branches indicate mitoses. Cells divide non-synchronously and cleavage patterns differ among embryos. In the fifth generation, some cells retard their cell cycle (arrows), marking the onset of differentiation. (D-F) Four-cell and post-gastrula stages. An asterisk indicates the site of polar body extrusion (position of the spindle) upon first cleavage of the embryo. Small arrows indicate the polar body; pha marks the pharynx anlage. (G,H) Views are from the upper levels of the embryos at the 32-cell stage. Sister blastomeres are connected with lines. The descendants of the eight-cell stage embryo were coloured in Photoshop in order to visualize the cell clones. To colour code the cells in G-L, we determined the orientation of the axis at the gastrulation stage and then stained the cells and 3D-representations of nuclei (J-L) with SIMI°BioCell according to the following rules. The most anterior cell of the four-cell stage is red and its sister is pink; the most posterior cell is blue and its sister is green. At the eight-cell stage the ventral descendants were stained a darker tone than their dorsal sister cells. All 3D representations were then rotated in the same orientation (left, anterior; top, ventral surface) and `run' to the 122-cell stage as shown in J,K. The division angles and blastomere arrangements differ in all embryos. Scale bar: 20 µm in D-F.

View larger version (68K): [in a new window]   Fig. 1. Thulinia stephaniae development: embryo 1. (A) In the fertilized egg the nucleus (n) is localized near the site of extrusion of the polar body (pb). (B) The first division is equal and the polar body is localized at the border of the two blastomeres. (C) Four-cell stage embryo. (D) Eight-cell stage embryo, sister blastomeres visible in this focal plane are connected with a line. (E) 16-cell embryo. (F) 32-cell stage embryo. As in earlier stages, the blastomeres are indistinguishable and all cells have contact with the surface of the embryo. (G) 64-cell stage embryo. Cells acquire a pyramidal shape, the nuclei are positioned at the surface of the embryo (white arrow). Sometimes a small blastocoel (co) is visible (black arrowhead). (H) Embryo after gastrulation. Ectodermal cells form an epithelium (white arrows) around the inner cell mass (icm) consisting of PGCs, mesoderm and endoderm precursors. (I) Germ layer differentiation and organogenesis. The gastrulated endodermal blastomeres proliferate and differentiate into the pharynx (ph) and gut (g) anlage. Mesodermal bands (mb) are located to the left and right of the endoderm. (J) Limb bud and somite formation. During differentiation of the somites (s) the ectoderm forms segmental limb buds (white arrows). The third and fourth limb buds are not in focus. The pharynx differentiates further and the gut anlage (g) consists of small cells. (K) Embryo after elongation. The ectoderm forms a ventral fold (vf) from which NPCs later delaminate to form the ganglia. The gut is surrounded by mesodermal cells, which are derived from the somites. (L) Embryo shortly before movement starts. Gut cells are vacuolized and form internal reflecting granulae (white arrows). Scale bar: 20 µm.

View larger version (77K): [in a new window]   Fig. 3. Gastrulation of Thulinia stephaniae: embryo 2. (A) Ventral side of the embryo. Gastrulation initiates 9 hours after egg deposition. Posterior (arrow) of the embryo is towards the left. The PGCs migrate anteriorly through the prospective mouth opening into the embryo. (B) After 2 hours, the pores, which correspond to the prospective mouth (pm, blastopore) and anus (pan), become visible. (C) Cell lineage of the gastrulating blastomeres up to the seventh generation. The mesoderm (M, red branch) and endoderm (End, blue branch) precursors are descendants of different blastomeres. The sister cells of the PGCs (G, yellow branch) found different germ layers and the PGCs are specified in different generations. (D) Gastrulation is completed and the pores are closed (arrows). (E) The stomodaeum (stom) forms from ectodermal cells at the anterior pole (right arrow). The inconspicuous pore becomes the proctodaeum (proc, left arrow). (F) Three-dimensional reconstruction and fate map of the blastula at the 124-cell stage before onset of gastrulation. Ventral view of the anterior blastopore. Nine mesodermal precursors (mes) are represented as red spheres. The pair of PGCs (yellow spheres) is surrounded by the germ layer precursors, including four endodermal (end) precursors (blue spheres). The remaining blastomeres acquire an ectodermal fate. Scale bar: 20 µm.

View larger version (115K): [in a new window]   Fig. 4. Mesoderm development in Thulinia stephaniae. The genealogy of a blastomere, which contributes to the first left limb bud somite, was traced back to the precursor blastomere (embryo 1). Dorsal view, anterior towards the left. (A) Position of the mesodermal precursor shortly before it immigrates during gastrulation (arrowhead). (B) Position of traced cell 2 hours later (arrowhead). The red spheres indicate the path of migration of the cell since immigration started (projected with the migration function of the SIMI°BioCell software). (C) After the next division, the cell (red sphere) touches the anterior inner side of the ectodermal epithelium. (D) Twenty hours after egg deposition, the mesodermal bands (mb) form out of the mesodermal precursors. The mesodermal bands (arrows) stretch from posterior to anterior along the pharynx (px) and gut anlagen. The red sphere marks the position of the traced cell. (E) Embryo 1.5 hours later. The mesodermal bands form somites left and right from the gut; no cavities can be seen (black arrows). The traced cell has divided once and participates in the formation of the left second somite. (F) Three-dimensional representation of the nuclei in the 122-cell stage embryo. The row of red spheres shows the path of migration of the traced mesoderm cell. (G) Positions of the mesoderm precursor during gastrulation. The white arrows indicate the position of the precursor during specified mitoses. Later, the cell migrates along the inner epithelium of the ectoderm. (H) Embryo 34 hours after egg deposition. Mesodermal cells proliferate into the limb buds (arrows). Scale bar: 20 µm.

View larger version (139K): [in a new window]   Fig. 5. Neurogenesis of Thulinia stephaniae: embryo 3. (A) Lateral view of an embryo that begins muscle contractions. The brain (br) and the ventral ganglia (I-IV) are highlighted in yellow. (B) Earlier lateral view of the same embryo after delamination of the neuronal precursor cells (arrows) and during formation of the ganglia. (C) Lateral view 15 hours earlier. After two cell divisions, the NPC are formed from the precursors. They are located in the ectodermal ventral fold inferior the future position of the ganglia. (D) Earlier ventral view. The arrows indicate the location of the NPCs in the 248-cell embryo. (E) Three-dimensional representation of the positions of the nuclei at this stage. The bright yellow spheres correspond to the NPCs, which are connected with white bars to their sister cells. Colour code see Fig. 2. The NPCs are descendants of both blastomeres of the two-cell stage. (F) View of the left side of the embryo after it has rotated at 28 hours. We followed one brain precursor (arrow) delaminating from the anterior dorsal ectoderm to start brain formation, prior to the delaminating NPCs, which form the ganglia. Scale bar: 20 µm.

View larger version (90K): [in a new window]   Fig. 6. Development of the germ cells in Thulinia stephaniae: embryo 3 (A) Three-dimensional representations of the positions of the primordial germ cells (PGC) (yellow spheres, arrows) after differentiation has occurred (last cell division). The neighbouring cells have large nuclei and are located at the site of the future blastopore. (B) The PGCs gastrulate (arrows). (C) The cell lineage analysis shows that the PGCs differentiated after the sixth cell division and are derived from both blastomeres of the two-cell stage. (D) Ventral view of the embryo, anterior towards the left. The PGCs are located posterior to the pharynx (px) and ventral to the midgut anlage. (E) Projection of the migration paths of the PGCs using SIMI°BioCell. During elongation of the embryo, the PGCs begin to migrate along independent paths to the prospective position of the gonad (left PGC green, right PGC red). (F) Final position of the PGCs (arrow) at the location where the gonad develops. Scale bar: 20 µm.

View larger version (91K): [in a new window]   Fig. 10. Juveniles formed after cell ablations in the embryo. (A-C) The same exuvia. (A) Three embryos were ablated. Two control embryos are marked with `c'. In the embryo (4-cell p) a polar blastomere and in the embryo labelled (4-cell s) a lateral blastomere have been ablated in the four-cell stage. In the third embryo (2-cell), one blastomere was ablated in the two-cell embryo. (B) Two empty eggshells (arrowheads) indicate that two juveniles have already hatched. (C) All juveniles have hatched from the exuvia and are in an anoxybiotic state. The smallest juvenile is derived from the cell ablation at the two-cell stage (arrow). (D) Embryos from a different ablation experiment at higher magnification. Left, a control embryo; right, an embryo after one cell was ablated in the two-cell stage. The stylet (white arrowhead) of the ablated embryo is reduced in comparison with that of the control embryo. Scale bars: A,B,C, 50 µm; D, 25 µm..

View larger version (90K): [in a new window]   Fig. 7. Development after laser ablation of one blastomere in the two-cell embryo. (A) 64-cell embryo. The ablated cell cleaved twice before its descendants degraded into small cytoplasts (star). The untreated blastomeres divided normally and the descendants acquire a pyramidal shape at the 124-cell stage (arrows). (B) Three-dimensional representation of the embryo shown in A. The non-ablated descendants first surround the remaining cytoplasts (yellow spheres) of the ablated cell, but are later found further from them. (C) Lineage of the embryo up to the 64-cell stage. The non-treated blastomeres undergo normal cell cycles. (D) Later stage of the embryo. The descendants of the non-treated blastomere form an epithelium (arrows) at the border of the cytoplasts (cp). (E) Later stage of the recorded embryo. A pair of PGCs is present and the embryo develops the main organs e.g. pharynx (px). The small embryo is surrounded by the cytoplasts. (F) Stage of limb bud (lb) formation. Anterior is towards the right. The cells of the ectoderm (arrows) are much larger than those found in normal embryos at the same stage. Scale bar: 20 µm.

View larger version (91K): [in a new window]   Fig. 8. Development after laser ablation of one cell in the four-cell stage embryo. (A) Forty-seven descendants of the untreated blastomeres surround (arrows) the ablated cell (x), which has divided once. The descendents degrade later into small cytoplasts. (B) Three-dimensional representation of the embryo shown in A, the yellow spheres mark the position of the two descendants of the ablated cell. The colour code is the same used in C. (C) Lineage of the 47-cell stage. As in a normal embryo of the corresponding stage (64-cell stage), some cell cycles are retarded. (D) Later stage after degradation of the ablated cell. The descendants of the living cells form a typical epithelium, and surround an inner endodermal cell mass (icm). The cytoplasts (cp) of the ablated cell are excluded. Two PGCs are found at the normal position (arrows). (E) Embryo before hatching. Mouth (mt), brain (br) and claws are present and look normal. The cytoplasts (cp) of the ablated cell surround the embryo. (F) Same stage as E under a higher magnification. The stylet (st), buccal tube and pharynx (px) are formed normally. The gut cells (gt) contain the typical granulae. Scale bar: 20 µm.

View larger version (91K): [in a new window]   Fig. 9. Development after ablation of two cousin blastomeres during the four-cell stage. (A) Lineage of the remaining two untreated blastomeres. The central lineage reflects the behaviour of the ablated blastomeres. The untreated blastomeres divide normally and show retarded cell cycles during the fifth generation (arrow). (B) Embryo at the 59-cell stage. (C) Three-dimensional representation of the nuclei positions of B. The descendants of the untreated blastomeres surround the ablated blastomeres. (D) Exuvia with the three embryos. In the embryos to the left and right, the ablated cells are marked with a cross. Sister cells are connected with a line. The embryo in the centre is a control embryo. (E) Later stage of D. Both treated embryos have a developed pharynx (white arrowheads). The control egg has already developed further. (F) Higher magnification of the left most embryo. Both PGCs are visible at the expected location for a normal embryo (black arrows). Scale bar: 20 µm.