Cell movement during chick primitive streak formation

Abstract

Gastrulation in amniotes begins with extensive re-arrangements of cells in the epiblast resulting in the formation of the primitive streak. We have developed a transfection method that enables us to transfect randomly distributed epiblast cells in the Stage XI-XIII chick blastoderms with GFP fusion proteins. This allows us to use time-lapse microscopy for detailed analysis of the movements and proliferation of epiblast cells during streak formation. Cells in the posterior two thirds of the embryo move in two striking counter-rotating flows that meet at the site of streak formation at the posterior end of the embryo. Cells divide during this rotational movement with a cell cycle time of 6-7 h. Daughter cells remain together, forming small clusters and as result of the flow patterns line up in the streak. Expression of the cyclin-dependent kinase inhibitor, P21/Waf inhibits cell division and severely limits embryo growth, but does not inhibit streak formation or associated flows. To investigate the role off cell-cell intercalation in streak formation we have inhibited the Wnt planar-polarity signalling pathway by expression of a dominant negative Wnt11 and a Dishevelled mutant Xdd1. Both treatments do not result in an inhibition of streak formation, but both severely affect extension of the embryo in later development. Likewise inhibition of myosin II which as been shown to drive cell-cell intercalation during Drosophila germ band extension, has no effect on streak formation, but also effectively blocks elongation after regression has started. These experiments make it unlikely that streak formation involves known cell-cell intercalation mechanisms. Expression of a dominant negative FGFR1c receptor construct as well as the soluble extracellular domain of the FGFR1c receptor both effectively block the cell movements associated with streak formation and mesoderm differentiation, showing the importance of FGF signalling in these processes.

Fig. 1

Transfection of cells in the early chick blastoderm results in predominant transfection of the epiblast. (A) Low magnification image of an embryo 5 h after the start of development and 3 h after transfection with pEGFP-N1 (Size bar 0.5 mm). (B) Confocal image of a section of the embryo shown in panel A where the actin cytoskeleton has been stained with rhodamine phalloidine (Size bar 50 μm). (C) Higher magnification image of same embryo shown in panels A, B. (Size bar 10 μm). (D) Section through the embryo shown in panels A–C. (E) Confocal image of section of embryo shown in panels A–D showing clear staining of cells in the upper epiblast and no labeled cells in the lower hypoblast layer. All embryos in this and subsequent figures are shown with the epiblast side on top. The embryo is oriented along the anterior–posterior axis with Koller’s sickle (only faintly visible) at the bottom of the image (A).

Fig. 2

Transfected embryos and tracks of GFP labelled cells during streak formation. We transfected a pre-streak embryo with a GFP expression vector pEGFPN1 and tracked the movement of individual GFP-expressing cells as described in Materials and methods. (A–C) Bright-field image, fluorescence image and cell trace of the same embryo. The cell trace is calculated over a period of 4 h. The green part indicates the movement during the last hour. The images shown are taken 7 h after the start of development, 4 h after transfection. (Size bar in panel A is 0.5 mm). (D–F) The same images of the same embryo 15 h after the start of the experiment. (G–I) The same images of the same embryo 18 h after the start of the experiment. All images of embryos are taken looking upon the epiblast in this and all following figures. (For additional information on the dynamics of the process see movie 1). The embryos are oriented with the tip of the streak pointing to the top. The white arrow in panel G points towards the tip of the forming primitive streak.

Fig. 3

Expression of p21Cip/Waf does not inhibit streak formation. (A) Bright-field image of a control embryo 18 h after electroporation with a pEGFP-N1 expression construct at Stage XIII. (Size bar 0.5 mm). (B) Merged fluorescence/bright-field image of the same control embryo showing the distribution of GFP transfected cells. (C) Bright-field image of an embryo expressing the CDK inhibitor p21Cip/Waf, also 18 h after transfection of a stage XIII embryo. (D) Merged fluorescence/bright-field image of the same embryo shown in panel C. Expression of p21Cip/Waf substantially reduces embryo growth, compare control (A) to p21Cip/Waf-expressing embryo (C), but does not inhibit streak formation (image width 4 mm). The embryos are oriented with the tip of the streak pointing to the right.

Fig. 4

Expression of dominant-negative Wnt11 does not inhibit streak formation, but inhibits the anterior extension of the primitive streak. (A–C) Bright-field image, fluorescence image and cell tracks of cells expressing a dominant negative Wnt11 construct. Tracks are calculated over 4 h, 4 h after transfection at stage XIII. (Size bar in panel A is 0.5 mm). (D–I) Same images after 9 and 18 h from the beginning of the recording respectively. Long before streak formation becomes visible the initial characteristic counter-rotating vortices are visible. However, once the streak forms (G–I), it does not extend anteriorly, the streak elongates by adding more cells to its posterior end. This change in behaviour is reflected by the cell tracks pointing backwards towards the bottom of the streak (I), where they appear to join in the streak. The movement of the cells over the last hour is shown in green. (see movie 2). The white line in panel G indicates the position of the streak with the tip of the streak pointing slightly up and to the right.

Fig. 5

Expression of Xdd1 does not inhibit the primitive-streak formation. (A, B) Fluorescence and bright-field images of an embryo transfected with a GFP expression construct at stage XIII and photographed after 18 h of development (Size bar is 0.5 mm). (C, D) Fluorescence and bright-field images of an embryo transfected with an Xdd1 expression construct at stage XIII and photographed after 18 h of development. (E, F) Fluorescence and bright-field images of an embryo transfected with a GFP expression construct at stage HH3 and photographed after 18 h of development. (G, H) Fluorescence and bright-field images of an embryo transfected with an Xdd1 expression construct at stage HH3 and photographed after 18 h of development. (I, J) Bright field image and cell track (4 h) of an embryo transfected with Xdd1/pEGFP-N1. (K, L) Bright field image and cell track (4 h) of an embryo transfected with Xdd1/pEGFP-N1 6 h after the images shown in panels I, J (see movie 3 for dynamics). Expression of the dishevelled mutant Xdd1 lacking the Dix domain does not inhibit streak formation (A, C). However, later development is severely abnormal suggesting that Xdd1 does interfere with planar-polarity signalling during later development (E, G). The embryos are oriented with the tip of the streak/heads pointing to the right.

Fig. 6

Blebbistatin does not inhibit streak formation, but does inhibit regression. (A, B) Un-incubated embryos were put in EC culture on agar–albumin plates containing 5 μM blebbistatin and photographed after 24 (A) and 48 h (B) respectively. (Size bar in panel A = 0.5 mm). (C, D) Un-incubated embryos were put in EC culture on agar–albumin plates. The embryos were photographed after 24 h (B) and 48 h (D), respectively. Streak formation (A, C) is not affected by blebbistatin at the concentration used, but that later development (B, D) is severely disturbed, there is a strong defect in the elongation of the embryo resulting in few compacted somites and large heads. These results are typical for results obtained in all embryos (15) investigated. The embryos are oriented with the tip of the streak (A, C) and heads (B, D) pointing to the right.

Fig. 7

Inhibition of primitive-streak formation by a dominant-negative FGFR1c construct and a FGFR1c-Fc fragment. (A–C) Bright-field image, fluorescence image and cell tracks calculated during 4 h of development, beginning 5 h after the transfection of a stage XIII embryo. (see movie 4). (D–F) Same images as in (A–C) of the same embryo, 6 h later. (Size bar in panel A is 0.5 mm). Expressing a GFP tagged dnFGFR1 receptor inhibits primitive streak and vortices in the epiblast. The colour coding of the tracks is as described in Fig. 2. Images are 3.3 mm wide. (G–I) Embryo transfected after 2.5 h incubation with pEGFP-N1. The images were taken 5 h after transfection, (G) 21.5 h later (I) Brachyury expression in the same embryo shown in panel H as detected by in situ hybridisation with a dioxygenin labelled RNA probe. (J–L) Embryo transfected after 2.5 h incubation with a FGFR1c-Fc. The images were taken 5 h after transfection (J) 21.5 h later (K). (L) Brachyury expression in the same embryo shown in panel K as detected by in situ hybridisation with a dioxygenin labelled RNA probe, not the absence of Brachyury expression. Note the lack of expression in FGFR1c-Fc-expressing embryos. We only observed 3 small Brachyury-expressing streaks in 12 transfected embryos, while we observed 12 Brachyury-expressing streaks in 13 control embryos transfected with pEGFPN1. The embryos are oriented with the tip of the