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Keywords:

  • gastrulation;
  • multicellular rosette;
  • Nodal signal;
  • Polonaise movements;
  • prestreak chick epiblast

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

During axis formation in amniotes, posterior and lateral epiblast cells in the area pellucida undergo a counter-rotating movement along the midline to form primitive streak (Polonaise movements). Using chick blastoderms, we investigated the signaling involved in this cellular movement in epithelial-epiblast. In cultured posterior blastoderm explants from stage X to XI embryos, either Lefty1 or Cerberus-S inhibited initial migration of the explants on chamber slides. In vivo analysis showed that inhibition of Nodal signaling by Lefty1 affected the movement of DiI-marked epiblast cells prior to the formation of primitive streak. In Lefty1-treated embryos without a primitive streak, Brachyury expression showed a patchy distribution. However, SU5402 did not affect the movement of DiI-marked epiblast cells. Multi-cellular rosette, which is thought to be involved in epithelial morphogenesis, was found predominantly in the posterior half of the epiblast, and Lefty1 inhibited the formation of rosettes. Three-dimensional reconstruction showed two types of rosette, one with a protruding cell, the other with a ventral hollow. Our results suggest that Nodal signaling may have a pivotal role in the morphogenetic movements of epithelial epiblast including Polonaise movements and formation of multi-cellular rosette.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

During early development in amniotes, the epithelial epiblast undergoes a variety of dynamic morphological changes, such as cell–cell division, ingression, migration, intercalation, and elongation of the primitive streak. During gastrulation, mesendoderm mesenchymal cells migrate away from the primitive streak to form three germ layers. In the early avian blastoderm, immediately after being laid, the embryo consists of a flat epithelial disk, in which the inner area pellucida and peripheral area opaca are distinguishable. Beneath the area pellucida, there are islands of cells, from which the hypoblast develops at the posterior margin of the area pellucida, eventually covering the area pellucida immediately before gastrulation (Kochav et al. 1980; Stern 2004). At the onset of/during primitive streak formation, large bilaterally symmetrical movements of epiblast cells toward the posterior region of the area pellucida occur in association with anterior movements of cells at its posterior midline (Cui et al. 2005). This large-scale tissue flow is called “Polonaise movements”, during which epiblast cells move in two striking counter rotating streams, which merge at the site of streak formation (Gräper 1929; Spratt 1946; Vakaet 1970; Chuai & Weijer 2008). Soon after the hypoblast has completely formed, a triangular shaped initial primitive streak develops in the caudal third of the area pellucida (Lawson & Schoenwolf 2001a). The initial primitive streak converges towards the midline and extends anteriorly to form a rod-shaped overt primitive streak, where large numbers of epiblast cells ingress to generate the mesendoderm mesenchyme. Fate map analysis of prestreak blastoderms showed that the prospective dorsal mesoderm is found in the posterior area pellucida adjacent to the sickle in the early blastula stage and then moves toward the center of the blastoderm in the late blastula stage (Hatada & Stern 1994). Such cellular movements appear to be caused by convergence of the posterior lateral epiblast toward the posterior midpoint and extension towards the center of the area pellucida along the midline and are consistent with “Polonaise movements” (Voiculescu et al. 2007).

During this large-scale counter-rotating cellular movement (Polonaise movements) in the prestreak epiblast, cells move within the simple columnar epithelial-epiblast, in which the cells are connected to each other via cadherin-based cell–cell contacts. Several cellular mechanisms have been proposed to regulate this peculiar cell movement during the initial formation of the primitive streak. The first proposal is that the precursor cells of the primitive streak undergo polarized cell division oriented perpendicular to the anteroposterior embryonic axis in order to extend the primitive streak (Wei & Mikawa 2000). The second suggests that convergent extension movements of the cells in the area pellucida of the posterior epiblast contribute to the formation of the initial primitive streak (Lawson & Schoenwolf 2001b). The third involves mediolateral intercalation of the posterior epiblast cells and their subsequent extension, with the intercalation/extension displacing lateral epiblast cells toward the midline (Voiculescu et al. 2007). Multicellular rosettes, which are proposed to link local cell intercalation to global tissue organization (Blankenship et al. 2006), are found in the primitive streak region of the epiblast (Chuai & Weijer 2008). All of these scenarios are plausible; however, it remains uncertain how epiblast cells move in the epithelial epiblast to form the primitive streak and how Polonaise movements are regulated.

The reciprocal tissue interactions that occur between the epiblast and hypoblast to initiate gastrulation movements have long been investigated. Waddington (1933) reported that the rotation of the lower layer of the avian blastoderm causes ectopic primitive streak induction, while recent experiments showed that rotation of the hypoblast (hypoblast + newly formed lower layer = endoblast) affects cellular movement in the adjacent epiblast, causing the streak to bend (Khaner 1995; Foley et al. 2000). Another experiment showed that hypoblast removal causes multiple streaks to form and that the displacement of Cerberus (a Nodal antagonist)-expressing hypoblasts away from the posterior margin of the area pellucida by the endoblast is necessary to initiate primitive streak induction (Bertocchini & Stern 2002). Activin, another member of the transforming growth factor-β (TGF-β) family, induces the formation of the ectopic primitive streak in the avian blastoderm (Ziv et al. 1992). Mouse embryos lacking Nodal activity arrest shortly before gastrulation and fail to form a primitive streak (Conlon et al. 1994). It is well known that in Xenopus, mice, and zebrafish, disruption of fibroblast growth factor (FGF) signaling strongly affects body axis formation and mesoderm induction (review, Böttcher & Niehrs 2005). In Xenopus embryos, FGF signals regulate gastrulation-related cell movements and morphology through a neurotrophin receptor homologue (Chung et al. 2005). In chick gastrulation, cell movement patterns from the streak are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8 (Yang et al. 2002; Chuai et al. 2006). In mouse FGFR-1 mutants show that the FGFR1 signal plays roles in directing mesoderm cell migration out of the primitive streak and its patterning during gastrulation (Deng et al. 1994; Yamaguchi et al. 1994). It was also reported that targeted disruption of FGF8 causes cell migration defects in gastrulating mouse embryos (Sun et al. 1999). These observations suggest that the signaling involved in mesoderm induction plays a role in the regulation of epiblast cell movements during streak formation in amniotes. However, little is known about not only the signaling but also cellular mechanisms responsible for the epiblast movements that precede primitive streak formation in amniotes.

Using chick blastoderm explantation experiments and DiI-cell-marking in whole embryo cultured blastoderm, we examined whether Nodal signaling is involved in morphogenetic movement of epiblast. We also examined the spatiotemporal restriction of multi-cellular rosettes in the epiblast layer and their three-dimensional structure.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Culture procedures

Fertilized eggs (Gallus gallus) were purchased from Shiroyama Farm (Kanagawa, Japan). The embryos were collected on phosphate-buffered saline (PBS) and staged according to Eyal-Giladi & Kochav (1976) and Hamburger & Hamilton (1951). Stage X–XI embryos (incubation = 0 h) were selected carefully, the posterior quarter of the area pellucida containing the sickle was extirpated and cut along the AP axis with a sharp tungsten needle, and cultured on chamber slides (Nunc) supplemented with serum-free medium (75% Dulbecco’s modified Eagle’s medium [DMEM], 25% McCoy’s medium, 10−7 mol/L dexamethasone, and penicillin-streptomycin, Matsui et al. 2008) or under various other test conditions, such as in medium with recombinant Lefty1 (R&D), Cerberus-S, or SU5402 (CALBIOCHEM). The point of explantation was marked, and the migrated distance between the explanted point and the center of the migrated explant was measured every 10 h (total incubation, 40 h) under an inverted microscope. The center of explant was defined by Lumina Vision (MITANI, Tokyo).

For making conditioned medium containing Cerberus-S, the COS-7 cells were transiently transfected with Xenopus Cerberus-S (a kind gift from Dr De Robertis, Piccolo et al. 1999) or pCS2 (+) using LIPOFECTAMINE 2000 (Invitrogen). After 24 h, the medium was replaced with serum-free DMEM (Sigma), the cells were cultured for an additional 48 h, and the conditioned medium was harvested. The resultant conditioned medium containing Cerberus-S was concentrated with centrifugal filters (cut-off MW = 10 KD; AMICON) and used.

Whole embryo culture and DiI injection

Stage X–XI embryos were selected carefully, washed in Tyroide’s solution, and subjected to DiI cell marking experiments according to the methods of Darnell et al. (2000) and Redkar et al. (2001). A stock solution of DiI (Molecular Probes) was prepared in dimethylsulfoxide (DMSO) (2.5 mg/mL) or absolute ethanol containing 10% glycerol (5 mg/mL). A freshly prepared working solution (1:200–500) in PBS was injected into the dorsal epiblast layer of the area pellucida at the 3, 6, and 9 o’clock positions (Fig. 2) using pulled glass needles (20 μm in external diameter) equipped with a pressure injector (60 psi, 10 ms; NARISHIGE). Subsequently, the embryos were placed on a vitelline membrane supported with a filter paper ring and processed for whole embryo culturing (EC-culture; Chapman et al. 2001). The embryos were cultured ventral side up on 500 μL of albumin-agar gel prepared in a 4-well dish (Nunc), and 50 μL of Tyroide’s solution were added with or without test reagent or concentrated conditioned medium containing Cerberus-S. Before and after 5 and 9 h of incubation (when the embryos had reached stage XII and XIV, respectively), DiI marked cells in the epiblast layer were observed and photographed under a fluorescent inverted microscope (KEYENCE). The triangular area made up of the three points (3, 6, and 9 o’clock) was calculated before and after incubation and then compared. Statistical analysis was performed using the paired t-test, and the significance level was set at <5%.

image

Figure 2.  rLefty1 affects epiblast cell movements during the blastula stage. Stage X–XI embryos were collected, DiI was pressure injected into the margin of the area pellucida at the 3, 6, and 9 o’clock sites, and then the embryos were EC-cultured. After 5 h (stage XII) and 9 h (stage XIV), the embryos were examined for DiI-marked cells under a fluorescent microscope. In the control and the embryos treated with 100 ng/mL rLefty1, the marked epiblast cells at 3 and 9 o’clock moved posteriorly, and the cells at 6 o’clock moved anteriorly; therefore, the triangular area composed of these three points was reduced significantly (Relative area). The DiI-marked cells in the embryos treated with 500 or 1000 ng/mL of rLefty1 failed to move; thus, there was no significant difference in the area of the triangle between before and after incubation. In embryos treated with 20 μmol/L SU5402, the DiI-marked cells moved in a similar manner to the control embryos. *P < 0.05; **P < 0.01; NS, no significant difference.

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In situ hybridization

Digoxigenin (DIG)-labeled single-stranded RNA was prepared using a DIG RNA labeling kit (Roche). cDNA of chick Brachyury (nucleotide positions 664–1140, NM_204940; Muhr et al. 1999) subcloned into the pGEM-T vector (Promega) was linearized using SacII and transcribed using SP6 RNA polymerase (Roche). Whole-mount in situ hybridization was performed as described by Nieto et al. (1996). The embryos were fixed in 4% paraformaldehyde in PBS for 2 h, washed with PBT (PBS containing 0.1% Triton X-100), dehydrated, and rehydrated through a graded series of methanol in PBT, before being fixed with 0.2% glutaraldehyde/4% paraformaldehyde in PBT for 20 min. After rinsing the samples with PBT, they were prehybridized with hybridization buffer (50% formamide, 5 × standard saline citrate [SSC] [pH 5.0], 50 μg/mL yeast tRNA, 1% sodium dodecyl sulfate [SDS], and 50 μg/mL heparin) for 2 h at 60°C and then hybridized with a digoxigenin-labeled probe (0.5 μg/mL in hybridization buffer) for 12 h at 60°C. After hybridization, the samples were washed in 2 × SSC for 1 h at 60°C and then in 0.2 × SSC for 1 h at 60°C. After rinsing the embryos with KTBT (Tris-buffered saline containing 1% Triton X-100), they were blocked with 20% sheep serum in KTBT for 1 h and then incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody for 12 h at 4°C. Hybridization was detected using 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium chloride (BCIP/NBT).

Reverse transcription–polymerase chain reaction

RNA was extracted from EC-cultured embryos or cultured explants as described previously (at least five embryos/explants were used in each experiment; Matsui et al. 2008). cDNA were synthesized from 0.2 μg of total RNA, and the polymerase chain reaction (PCR) was carried out in 10 μL of reaction buffer (QIAGEN). The primers for Brachyury and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were described elsewhere (Matsui et al. 2008). The samples were cycled at 94°C for 30 s at an annealing temperature of 55°C and then at 72°C for 1 min, with final extension being performed at 72°C for 10 min. The number of cycles required for each primer was as follows: Brachyury: 26 and GAPDH: 23.

Fluorescent microscopy

Stage X–XIII embryos were fixed in 4% paraformaldehyde in PBS, washed in PBS, and stained with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin and 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI) for 30 min at room temperature. After an extensive wash, the embryos were transferred to slides and coverslipped, and the samples were observed under a laser confocal microscope (Zeiss, Leica). The number of multicellular rosettes, in which more than six cells shared a central cellular junction (Blankenship et al. 2006), was counted in 0.068 mm2 epiblast regions in the anterior, posterior, left, right, and central regions of the area pellucida and was compared between regions. Statistical analysis was performed using the t-test, and the significance level was set at <5%.

Three-dimensional rosette reconstruction was performed as follows. Epiblast regions, in which the rosettes were contained, were scanned by laser confocal microscopy every 0.5 μm, and 41 sections (20 μm in thickness) were obtained. Using Photoshop (Adobe), the cells that made up the rosette were selectively traced, and the resultant 41 sections containing a rosette were reconstructed three-dimensionally with Delta Viewer (http://delta.math.sci.osaka-u.ac.jp/DeltaViewer/index-j.html).

For TRITC-phalloidin staining for tissue sections, embryos were fixed in 4% paraformaldehyde in PBS, embedded in OCT compound, and frozen sections were cut on a cryostat. Sections were stained with TRITC-conjugated phalloidin and DAPI.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Inhibition of Nodal signaling affects epiblast movement

First, we screened whether Nodal and/or FGF signaling was able to regulate the epiblast movements in cultured posterior blastoderm, in which epiblast cells can move on the chamber slide and differentiate to express stage-dependent marker genes. The posterior quarters of the blastoderms from stage X to XI embryos were cultured on chamber slides supplemented with serum-free medium with or without rLefty1, Cerberus-S, or SU5402. The Lefty proteins antagonize Nodal signaling through its interaction with EGF-CFC as well as Nodal ligand but not Activin (Shen 2007). Cerberus-S blocks Nodal signaling via its direct interaction with Nodal ligand but not BMP4/Wnt8 (Piccolo et al. 1999). The resultant explants were examined to assess the distance (whole tissue movement) migrated from the point of explantation every 10 h. As shown in Figures 1A,B and S1, the migration of explants treated with Lefty1 reduced in a dose dependent fashion and was suppressed significantly at 10, 20, 30, and 40 h when cultured in medium containing with 1 μg/mL Lefty1 (P < 0.01, Figs 1B, S1). In addition, explants cultured in Cerberus-S-containing conditioned medium failed to migrate (Figs 1C, S1). The explants treated with SU5402 (5 μmol/L) migrated similarly to the control explants until 20 h; however, the migration of the explants treated with SU5402 decreased significantly at 30 and 40 h in culture (P < 0.01, Fig. 1D). The resultant cultures were subjected to reverse transcription–polymerase chain reaction (RT–PCR) to detect mesoderm marker Brachyury, and showed that the PCR products of Brachyury were detected when the explants were treated with a Nodal antagonist, but not detectable in explants treated with SU5402 after 40 h in culture (Fig. 1E). Results indicated that the inhibition of Nodal activity affected the initial migration of explants until 20 h, at which Brachyury mRNA begins to be detectable by RT–PCR (indicating streak stage), suggesting Nodal signaling may have a role in the migration of epiblast cells during Polonaise movements.

image

Figure 1.  Anti-Nodal properties inhibit the initial migration of cultured explants. The posterior quarters of the blastoderms from stage X to XI embryos were cultured on chamber slides supplemented with serum-free medium with or without rLefty1, medium containing Cerberus-S, Mock conditioned medium, or SU5042. The resultant cultures were examined to assess the distance migrated from the point of explantation every 10 h. The migration of explants treated with rLefty1 was reduced (A, B). In addition, explants cultured in concentrated conditioned medium containing Cerberus-S (the final concentration ratio was 20×) failed to migrate (C). Explants treated with SU5402 migrated similarly to control explants up to 20 h; however, the migration was decreased significantly at 30 and 40 h in culture (D). The resultant cultures (total incubation, 40 h) were subjected to reverse transcription–polymerase chain reaction (RT–PCR) to detect Brachyury and the results showed that the PCR products of Brachyury were detected even when the explants were treated with a Nodal antagonist (E), but not detectable in explants treated with SU5402. Cer. S, Cerberus S; *P < 0.05; **P < 0.01.

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To examine the role of Nodal in epiblast movements in vivo, DiI-cell-marking was attempted in whole embryo-cultured blastoderm (EC-culture, Chapman et al. 2001). Stage X–XI embryos were collected, and DiI was pressure injected into the margin of the area pellucida at 3, 6, and 9 o’clock sites (Fig. 2). After an appropriate incubation period, when the embryos had reached stage XII (5 h in culture) or stage XIII–XIV (9 h), they were examined for the migration of DiI-marked cells under an inverted fluorescent microscope. When the embryos were cultured in Tyroide’s solution alone, the marked cells at 9 and 3 o’clock moved toward the posterior region of the epiblast, and the cells at 6 o’clock had moved anteriorly after 5 and 9 h incubation, thereby reducing the triangular area made of these three points significantly (Relative area in Fig. 2). On the other hand, the DiI-marked cells in the embryos treated with 500 or 1000 ng/mL rLefty1 failed to move (Fig. 2). In embryos treated with rLefty1 (500 or 1000 ng/mL), there was no significant difference in the area of the triangle between the onset of incubation and after 5 or 9 h incubation (Fig. 2). Conditioned medium containing Cerberus-S failed to inhibit the migration of DiI-marked cells in EC-culture (not shown). In embryos treated with 20 μmol/L of SU5402, DiI-marked cells were able to move in a similar manner to those observed in the control embryos (Fig. 2). After additional 5 h incubation, the control embryos (89%, 24/27) and embryos treated with 100 ng/mL of rLefty1 (73%, 11/15) had formed an overt primitive streak, in which the expression of Brachyury was detected by whole mount in situ hybridization (PS in Fig. 3). On the other hand, embryos treated with 500 or 1000 ng/mL rLefty1 failed to form primitive streak (25%, 4/16 and 4%, 2/25, respectively; Fig. 3), and the incidence of Brachyury expression was reduced (41%, 5/12 and 24%, 5/21, respectively; Fig. 3). Interestingly, some embryos treated with 500–1000 ng/mL of Lefty1 failed to form a primitive streak but expressed Brachyury in the posterolateral region of the area pellucida with a patchy staining pattern (arrowheads in Fig. 3). In similarly cultured embryos, RT–PCR showed that the PCR products of Brachyury were detectable in embryos treated with rLefty1 but that the PCR products of Brachyury were reduced in embryos treated with Lefty1 in a dose dependent fashion. In embryos treated with 10–20 μmol/L SU5402, primitive streak was formed; however, the width of streak was narrow and the expression of Bracyury was reduced (Fig. 3). BrdU incorporation in the cultured blastoderms showed that the mitotic index in the embryos treated with 1000 ng/mL rLefty1 was similar to that in the control cultures (not shown). In addition, no significant increase in apoptosis was observed in the embryos treated with rLefty1 (not shown). These observations indicated that the inhibition of Nodal signaling by Lefty1 affected epiblast cell movement at the early to late blastula stages during which Polonaise movements occur.

image

Figure 3.  rLefty1 inhibits the formation of the primitive streak. Stage X–XI embryos were EC-cultured with or without rLefty1. After 14 h (total incubation time; the control embryos had reached stage 3), the embryos were subjected to whole mount in situ hybridization or reverse transcription–polymerase chain reaction (RT–PCR) to detect the mesoderm marker Brachyury. The control embryos and the embryos treated with 100 ng/mL of Lefty1 formed an overt primitive streak (PS), in which the expression of Brachyury was detected. The embryos treated with 500 or 1000 ng/mL rLefty1 failed to form PS. In situ hybridization showed that the expression of Brachyury in the PS was apparent in the control and embryos treated with 100 ng/mL of Lefty1. In the embryos treated with 500 or 1000 ng/mL Lefty1, the percentage expression of Brachyury was reduced. Although, embryos treated with 500–1000 ng/mL of Lefty1 failed to form PS, some embryos expressed Brachyury in the posterolateral region of the area pellucida with patchy staining (arrowheads). In embryos treated with SU5402, primitive streak was formed but the width of streak was narrow and the expression of Brachyury was reduced. RT–PCR was performed and the results showed that the PCR products of Brachyury were detectable but that their expression was reduced in embryos treated with Lefty1 or SU5402 in a dose dependent manner.

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Rosette formation is predominant in the posterior half of epiblast at stage XII

The above experiments suggested that the Polonaise movements are controlled, at least in part, by Nodal signaling during the early to late blastula stages. Multi-cellular rosette formation has been observed in Drosophila epithelial cells undergoing cell–cell intercalation during germ band extension (Blankenship et al. 2006) and in the epiblast/ectoderm during chick gastrulation (Bancroft & Bellairs 1974; Chuai & Weijer 2008; Wagstaff et al. 2008). We therefore examined the spatiotemporal density of rosettes and whether Nodal antagonism affected the formation of rosettes in the pregastrula epiblast. Stage X, XII, and XIII embryos were stained with TRITC-phalloidin, and we counted the rosettes in 0.068 mm2 epiblast regions in the anterior (ant), posterior (post), left, right, and central regions of the area pellucida (Fig. 4). At stage X, the number of rosettes was <5/0.068 mm2 in all regions, and there was no significant difference in the number of rosettes between regions (Fig. 4A,B). At stage XII, the number of rosettes in the posterior, right, and central regions was significantly increased compared with that in the anterior region (P < 0.05, Fig. 4B). At stage XIII, each epiblast cell was smaller than at stage X, and the number of rosettes in each region was increased (Fig. 4A,B). There was no significant difference in the number of rosettes between the regions at stage XIII (Fig. 4B). Our results indicated that the number of rosettes significantly increases in the posterior half of the stage XII epiblast, including left, right, center and posterior regions, in which Polonaise movements occur and a sheet-like hypoblast begins to be apparent beneath the epiblast (Eyal-Giladi & Kochav 1976).

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Figure 4.  Multicellular rosettes are predominantly seen in the posterior half of epiblast at stage XII. Stage X, XII, and XIII embryos were stained with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin, and the number of rosettes in 0.068 mm2 epiblast regions in the anterior (ant), posterior (post), left, right, and central regions of the area pellucida was counted. The density of rosettes (yellow dots in A) in stage X posterior epiblast was sparse in comparison with that seen in stage XIII posterior epiblast; and the cells in the stage XIII epiblast were smaller than stage X (A). The number of rosettes was <5/0.068 mm2 in all regions at stage X, whereas at stage XII the number of rosettes in the posterior (post), right, and central regions was significantly increased compared with that in the anterior (ant) region (P < 0.05, B). At stage XIII, the number of rosettes in each region was increased and there was no significant difference in the number of rosettes between the regions. *P < 0.05.

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We next attempted three-dimensional reconstruction of the rosettes. Stage X and XII embryos were stained with TRITC-phalloidin and DAPI, scanned using a confocal microscope, and reconstructed (five rosettes at stage X and eight rosettes at stage XII were examined). In one type of rosette (4/5 at stage X and 4/8 at stage XII, Fig. 5 upper panels), the cells constituting the rosette came into contact with each other at the center of the rosette on the dorsal surface, while on the ventral surface, no rosette structure was evident because one cell that was not visible on the dorsal surface was seen (* in Fig. 5 upper panels). In the other type of rosette (1/5 at stage X and 4/8 at stage XII, Fig. 5 lower panels), all cells that made up the rosette came into contact with each other at the center of the rosette on the dorsal and ventral surfaces, and there was a small hollow (arrows in Fig. 5 lower panels) on the ventral surface of the epiblast. Our observations indicated that the number of rosettes is significantly higher in the posterior half of the epiblast at stage XII and that there are at least two types of rosette in the pre-streak epiblast.

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Figure 5.  Three-dimensional observation of multicellular rosettes. Stage X and XII embryos were stained with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin (green) and 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI) (purple) and scanned using a confocal microscope. The cells constituting rosettes were traced and reconstructed. The reconstructed 3D-rosette was cut vertically along the yellow broken line and observed from the side, dorsal, and ventral oblique directions. The upper panels show one type of rosette containing a ventral cell (4/5 at stage X, 4/8 at stage XII). In this type, the cells constituting the rosette come into contact with each other at the dorsal surface, while no rosette structure is evident on the ventral surface because one cell (*), which is not visible on the dorsal surface, lies at the center of the rosette. The lower panels show another type of rosette (1/5 at stage X, 4/8 at stage XII), in which a ventral hollow (arrows) is observed.

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Inhibition of Nodal signaling affects rosette formation

We next examined whether a Nodal antagonist could affect rosette formation in EC-cultured embryos. Stage X–XI blastoderms were cultured in medium supplemented with or without rLefty1 or SU5402. After an appropriate incubation period, when the embryos had reached stage XIII–XIV, they were examined to assess the number of rosettes they contained. As shown in Figure 6, when the embryos were treated with rLefty1 (100–1000 ng/mL), the number of rosettes was significantly reduced compared with that in the control cultures in a dose dependent manner. In the embryos treated with SU5402 (1–5 μmol/L), the number of rosettes was not affected (Fig. 6). Another experiment showed that Lefty1-soaked bead implanted at the posterior epiblast at 6 o’clock affected not only the cellular movement but also the formation of rosettes surrounding the Lefty1-bead (Fig. S2). We next examined morphological differences of epiblast layer between the control embryos and embryos treated with Lefty1 in histological sections that were stained with TRITC-phalloidin (Fig. 7). Results showed that control epiblast consisted of tall epiblast cells with accumulation of phalloidin at apical regions, whereas in embryos treated with Lefty1 epiblast layer was thinner and consisted of round and short cells with weak phalloidin staining at apical regions (Fig. 7). These results indicated that an inhibition of Nodal activity affected the cellular morphogenetic changes of the epiblast layer at prestreak stages.

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Figure 6.  rLefty1 affects rosette formation. Stage X–XI embryos were cultured in medium with or without rLefty1 or SU5402. After 9 h incubation (stage XIV), the embryos were examined for the number of rosettes they contained. When the embryos were treated with rLefty1 (100–1000 ng/mL), the number of rosettes was significantly reduced. In embryos treated with SU5402 (1–5 μmol/L), the number of rosettes was not affected.

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image

Figure 7.  rLefty1 affects epithelial morphology of epiblast layer. Stage X–XI embryos were cultured in medium with or without rLefty1. After 9 h, embryos were fixed, cryostat sections of posterior region were cut, and stained with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin and 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI) (nuclear staining). Results showed that control epiblast was a columnar epithelium with accumulation of phalloidin at the apical region of the epiblast; in contrast, the epiblast layer in embryos treated with Lefty1 appeared to be thinner and consisting of round and short cells with weak phalloidin staining at apical regions. epi, epiblast; hypo, hypoblast.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

In the present study, we showed that the inhibition of Nodal signaling affected the migration of DiI-marked epiblast cells during the early to late blastula stages. Interestingly, some Lefty1-treated streak defective embryos showed a patchy deposition of Brachyury in the posterior region of the epiblast, suggesting that prospective mesodermal cells that had failed to migrate expressed Brachyury. These observations suggest that Nodal signaling is required for the epiblast cell movements that regulate Polonaise movements before the streak formation. In mice possessing an insertional mutation related to Nodal activity, either the earliest sign of primitive streak formation (local thickening of the posterior epiblast) or the formation of the mesoderm layer is defective, suggesting that the Nodal signal is required for the local accumulation of cells that accompanies gastrulation (Conlon et al. 1994). They also reported that some embryos show randomly positioned patches of the posterior mesoderm suggesting that prospective mesoderm populations fail to migrate appropriately from their point of origin. ES cell chimera experiments have also shown that Nodal-deficient ES cells preferentially populate the anterior component of the epiblast, indicating that migratory defects exist in Nodal-deficient cells (Lu & Robertson 2004). Chimeric embryos derived from the injection of Alk4 (Nodal type I receptor)-null cells into the blastula showed that Alk4-null cells are capable of mesoderm formation but fail to form a primitive streak (Gu et al. 1998). In mice possessing mutant Smad2, a downstream signaling effector protein of Nodal/TGF-β, the primitive streak fails to form; thus, defective anterior posterior patterning of the embryo occurs, and prospective proximal epiblast cells are expanded (Waldrip et al. 1998). The nodal co-factor Cripto is also reported to be required for correct orientation of the anterior-posterior axis in mouse embryos (Ding et al. 1998). The phenotypic features seen in mutant/chimeric embryos with defective Nodal signaling are strikingly similar (i.e., defective primitive streak formation and dislocated mesoderm tissue), suggesting that Nodal signaling acts in an autocrine fashion to instruct directional epiblast cell movements toward the region of the streak. Taking our results together with those of others, it is strongly suggested that Nodal signaling may play a central role in the morphogenetic movements of epiblast cells (Polonaise movements) during the establishment of the primitive streak in amniotes.

During Polonaise movements, the posterior epiblast cells in the area pellucida move toward the center of the epiblast, and the cells in the lateral margin of the area pellucida move toward the posterior midline. The direction of the bilateral counter-rotating cellular movements, which merge at the site of streak formation, is regulated through the anterior posterior midline (Cui et al. 2005). During the early blastula stages in chick embryos, Nodal, the expression of which is thought to be induced by Wnt8c and cVg1, is expressed in epiblast cells located at the posterior lateral margin of the area pellucida (Lawson et al. 2001c; Skromne & Stern 2002). The expression pattern of Nodal is consistent with the epiblast region from which cells undergo Polonaise movements during stages XI–XII. However, it is uncertain how the direction of Polonaise movements is regulated and why they do not invade into the area opaca. In the early blastula, the hypoblast develops beneath the posterior epiblast, in which Polonaise movements occur. It has been reported that rotation of the hypoblast affects the direction of Polonaise movements and results in axis deviation (Khaner 1995; Foley et al. 2000; Voiculescu et al. 2007). Another experiment has shown that hypoblast removal causes ectopic expression of Nodal as well as multiple streaks to form as the hypoblast emits inhibitors of axis formation, such as Cerberus, and that newly formed sickle endoblasts that do not express Nodal antagonists dislocate the posterior hypoblast; therefore, the strongest Nodal signaling might occur in the posterior epiblast (Bertocchini & Stern 2002). In mice, Nodal antagonism induced by Cerberus and Lefty1 in the anterior visceral endoderm restricts primitive streak formation to the posterior end of mouse embryos (Perea-Gomez et al. 2002). In the chick blastula, the bone morphogenetic protein (BMP)-antagonist Chordin is expressed in the posterior area pellucida, and misexpression of BMP4 inhibits primitive streak formation; furthermore, BMP7 is expressed abundantly in the area opaca; therefore, it is suggested that the expression of BMP in the area opaca is likely to suppress the formation of the primitive streak (Streit et al. 1998). These observations suggest that Polonaise movements are controlled by autocrine positive signaling mediated by Nodal as well as inhibitory signals from the surrounding tissues (the hypoblast and area opaca).

In the present study, we showed that an inhibition of Nodal activity affected the epithelial morphology of the pre-streak epiblast, such as formation of rosette and epiblast cell shape in tissue section. Furthermore, accumulation of actin at the epiblast cortex was affected in embryos treated with Lefty1. Our preliminary experiment showed that ROCK1 expression was downregulated in epiblast treated with Lefty1 (not shown). Although the molecular mechanisms of mesendoderm induction are disclosed, signals controlling the cellular mechanisms that are required for gastrulation movements are largely unknown (Heisenberg & Solnica-Krezel 2008). It has been reported that actomyosin dependent cell cortex tension, regulated by Nodal/TGF-β signaling, directs progenitor cell sorting during gastrulation (Krieg et al. 2008). We previously reported that ROCK1/2 is expressed in gastrulating cells (Sakata et al. 2007), and TGF-β signal is able to upregulate the expression of ROCK1 in endocardial cells at the onset of epithelial-mesenchymal transition in valvuloseptal endocardial cushion formation (Sakabe et al. 2008). These observations suggest that Nodal signal is necessary to elicit the morphogenetic changes of epiblast cells via the cytoskeleton/actomyosin reorganization during Polonaise movements.

SU5402, an inhibitor of the tyrosine kinase activity of FGFR1 (Mohammadi et al. 1997), did not affect not only the migration of DiI-marked epiblast cells but also established the streak in vivo. FGFR1-null mice die at gastrulation but possess an established primitive streak and accumulate prospective mesoderm cells in the streak region (Yamaguchi et al. 1994; Deng et al. 1994). Chimeric analysis of FGFR1 function suggests that the FGFR1 signal regulates the epithelial-mesenchymal transition of the primitive streak in epiblasts and the subsequent morphogenetic movements of mesoderm cells through the streak by controlling Snail and E-cadherin levels (Ciruna et al. 1997; Ciruna & Rossant 2001). In FGF8-null mutant embryos, epiblast cells move into the streak and undergo epithelial-mesenchymal transition but fail to move away from the streak (Sun et al. 1999). In chick gastrulation, mesoderm cell migration from the streak is controlled by chemotaxis mediated by FGF signaling (Yang et al. 2002). Taken together with these observations, it is suggested that signaling mediated by FGFR1 is not likely to play a direct role in epiblast cell movement before the formation of the primitive streak in amniotes.

It has been reported that multicellular rosette formation in epithelial sheets is observed in several developmental processes, and it is thought to be involved in morphogenesis, such as in germ band extension in Drosophila and neural tube closure in chicks (Blankenship et al. 2006; Nishimura & Takeichi 2008). In our study, the density of rosettes in the epiblast was increased in the posterior lateral region at stage XII, where Polonaise movements and polyingression of epiblast cells occur, and a Nodal antagonist inhibited not only epiblast cell movements but also rosette formation. Furthermore, we found that there are at least two types of rosette, one is a rosette with a central cell protruding from its ventral surface (this central cell is not visible from the dorsal side) and predominant at earlier stage; the other is a rosette with a hollow on its ventral surface. This observation reminds us that some hypoblast cells originate from the epiblast; and thus, there are holes in the ventral surface of the epiblast through which prospective hypoblast cells undergo polyingression (Lawson & Schoenwolf 2001a). Therefore, it is possible that the protruding central cell seen in the ventral surface of the rosette is a future hypoblast cell, and the rosette with the ventral hollow may result from its ingression. The role of rosettes in Polonaise movements is uncertain, but it is possible that the ingression of epiblast cells is responsible for providing a space into which migrating epithelial cells can enter in the manner of a “sliding puzzle” during epithelial epiblast cell movements. According to our observations, we hypothesized that rosettes in the epithelial epiblast are responsible for the morphogenetic events that precede streak formation. However, it is largely uncertain whether rosettes are required for epithelial movement and if so, how they contribute to it because it is impossible to selectively perturb their formation.

We showed that Nodal signaling may have a role in the regulation of pre-streak epiblast morphogenesis including cellular movements and formation of rosette during Polonaise movements. Further experiments should be necessary to elucidate the cellular mechanisms regulating the morphogenesis in amniotes’ pregastrula epiblast.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

This work was supported by JSPS Grant-in-aid for Scientific Research #20390052. The authors thank Dr De Robertis for providing Cerberus-S DNA and M. Takahashi and S. Uoya for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Fig. S1. Anti-Nodal properties inhibit the initial migration of cultured posterior blastoderm explants.

Fig. S2. Lefty1-soaked bead affects epiblast cell movements and rosette formation.

FilenameFormatSizeDescription
DGD_1244_sm_f1.tif9039KSupporting info item
DGD_1244_sm_f2.tif5414KSupporting info item

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