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Developmental Biology 305 (2007) 161 – 171 www.elsevier.com/locate/ydbio Formation of the dorsal marginal zone in Xenopus laevis analyzed by time-lapse microscopic magnetic resonance imaging Cyrus Papan ⁎, Benoit Boulat, S. Sendhil Velan 1 , Scott E. Fraser, Russell E. Jacobs Beckman Institute, California Institute of Technology, 1200 California Blvd., Pasadena, CA 90124, USA Received for publication 11 December 2006; revised 16 January 2007; accepted 5 February 2007 Available online 13 February 2007 Abstract The dorsal marginal zone (DMZ) of the amphibian embryo is a key embryonic region involved in body axis organization and neural induction. Using time-lapse microscopic magnetic resonance imaging (MRI), we follow the pregastrula movements that lead to the formation of the DMZ of the stage 10 Xenopus embryo. 2D and 3D MRI time-lapse series reveal that pregastrular movements change the tissue architecture of the DMZ at earlier stages and in a different fashion than previously appreciated. Beginning at stage 9, epiboly of the animal cap moves tissue into the dorsal but not into the ventral marginal zone, resulting in an asymmetry between the dorsal and the ventral sides. Time-lapse imaging of labeled blastomeres shows that the animal cap tissue moves into the superficial DMZ overlying the deeper mesendoderm of the DMZ. The shearing of superficial tissue over the deeper mesendoderm creates the radial/vertical arrangement of ectoderm outside of mesendoderm within the DMZ, which is independent of involution and prior to the formation of the dorsal blastoporal lip. This tilting of the DMZ is distinct from, but occurs synchronously with, the vegetal rotation of the vegetal cell mass [R., Winklbauer, M., Schürfeld (1999). “Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus.” Development. 126, 3703–3713.]. We present a revised model of gastrulation movements in Xenopus laevis. © 2007 Elsevier Inc. All rights reserved. Keywords: Developmental biology; Xenopus laevis; Gastrula; Spemann organizer; Longitudinal study; MRI; Contrast media Introduction The dorsal marginal zone (DMZ) is a key embryonic tissue region of the early gastrula stage Xenopus embryo because it houses the Spemann–Mangold organizer (Spemann and Mangold, 2001). It contains precursors of all three germ layers (Keller, 1975, 1976); thus, knowledge of the cell movements and the timing of the cell interactions are important for understanding inductive processes among the cells within the DMZ (Nieuwkoop, 1999; Nieuwkoop and Koster, 1995). In the current view, Xenopus gastrulation begins with the appearance of a dorso-vegetal pigment line at stage 10, marking ⁎ Corresponding author. Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos 04-01, Singapore, 138669, Singapore. Fax: +65 6478 9080. E-mail address: [email protected] (C. Papan). 1 West Virginia University, Department of Radiology and Center for Advanced Imaging, One Medical Center Drive HSC South, PO Box 9236, Morgantown, WV 26506-9236, USA. 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.02.005 the site of the future blastopore lip (Keller and Davidson, 2004; Nieuwkoop and Faber, 1994; Shih and Keller, 1994). Following this, mesendodermal precursors are thought to be brought into vertical contact beneath the overlying ectoderm by means of two types of morphogenetic movements: involution (Keller, 1975; Vogt, 1929) and vegetal rotation (Winklbauer and Schürfeld, 1999), both of which are thought to begin at stage 10 (Poznanski and Keller, 1997; Shih and Keller, 1994; Winklbauer and Schürfeld, 1999). However, histological examination makes it likely that the first morphogenetic movements occur prior to stage 10, and internal tissue rearrangements before this stage have been described (Bauer et al., 1994; Brachet, 1935; Hausen and Riebesell, 1991; Holtfreter, 1943; Keller, 1978; Nieuwkoop and Florschütz, 1950; Schechtman, 1934; Vodicka and Gerhart, 1995; Vogt, 1929). Nevertheless, the true nature, timing and extent of these motions, and their relationship to the outward signs of gastrulation, have remained uncertain. Due to the optical opacity of amphibian embryos, hypotheses about 162 C. Papan et al. / Developmental Biology 305 (2007) 161–171 internal tissue movements have relied on the observation of live tissue explants, on interpretations of tissue movements from fixed material or on extrapolations from the externally visible cell movements. Thus, the morphogenetic tissue movements at blastula and early gastrula stages and their significance for tissue interrelationships have not been convincingly defined. Here we use 2D and 3D time-lapse microscopic magnetic resonance imaging (MRI) to trace the pregastrular tissue movements that result in the formation of the DMZ at stage 10. We find that the tissue movements leading to the formation of the DMZ begin by stage 9. Pregastrula epiboly moves tissue from the animal cap into the DMZ, but not into the ventral marginal zone, explaining the well-known dorso-ventral tissue asymmetry in the marginal zone of the stage 10 Xenopus embryo (Hausen and Riebesell, 1991; Nieuwkoop and Florschütz, 1950). Time-lapse tracing of B1- and C1-blastomeres labeled with MRI contrast agent confirms this process and reveals that animal cap cells (e.g. descendants of the B1-blastomere) form the superficial DMZ by moving outside mesendoderm cells (e.g. the descendants of the C1-blastomere). Thus, beginning at stage 9, pregastrula epiboly transforms the planar/horizontal tissue juxtaposition into radial/vertical tissue juxtaposition prior to the formation of the dorsal blastoporal lip. The outcome is the same as proposed to result from an internal involution movement; however, this apposition is not brought about by involution (the in-folding of invaginating tissue back onto an external cell layer). Time-lapse analysis furthermore reveals that the tissue movements associated with vegetal rotation (Winklbauer and Schürfeld, 1999) also begin at stage 9, much earlier than previously appreciated. These vegetal rotation motions bring a portion of the blastocoel floor into contact with the animal cap in the upper (anterior) deep marginal zone. Epiboly and vegetal rotation occur synchronously, so that by the onset of gastrulation, the vegetal cell mass has come into contact with the animal cap by two different paths. Based on these observations we present a schematic model of the pregastrula morphogenetic movements and DMZ formation. Materials and methods Embryo handling, mounting, imaging and image processing Embryos were handled, mounted and imaged as described previously (Papan et al., 2006, 2007). If embryos were to be injected with dye, they were de-jellied at the 4- to 8-cell stage by incubating them in 2% cystein pH 8.0 for 4– 5 min and rinsed with Rearing medium. For 3D imaging, the Rearing medium Fig. 1. 2D time series showing how animal cap tissue moves into the DMZ but not the VMZ during late blastula. Embryonic stages (Nieuwkoop and Faber, 1994) are given at the upper left. The elapsed time is shown at the lower right in hour:minute relative to the first scan shown. “Hist” shows an optical image of a stage 10.25 embryo for comparison. Images are sagittal, the dorsal side is to the right and the animal pole is up. A color look-up table (shown at the right of the figure) was applied, with the animal cap (AC) rendered in blue and the vegetal cell mass (veg) in yellow. The blastocoel (bc) is white. The black horizontal lines indicate the position of the animal cap boundaries at stage 8. Placing the same line on the subsequent images indicates the original position of the boundary. White lines indicate the moving front of the animal cap tissue in the subsequent stages. For confirmation of the stage, the inset figure in 10.25 shows the blastopore morphology of the embryo immediately after the scan. In “Hist”, the white dotted lines indicate the boundary between the region of smaller cells of the marginal zone and the larger cells of the vegetal cell mass. Arrowhead = dorsal blastoporal lip. Scale bar = 200 μm. C. Papan et al. / Developmental Biology 305 (2007) 161–171 was supplemented with Feridex (Berlex, Seattle, WA) at a 1:40 dilution to suppress the water signal. No morphological differences were observed in comparison to sibling embryos developing in a Petri dish. To slow development, scanning was done at 15 °C. At this temperature, 24 h are required to reach stage 12, yielding sufficient temporal resolution for 3D time series to follow development. For confocal microscopy, embryos were fixed in MEMFA and sagittally bisected with a razor blade in order to image just beneath the cut face. Embryos were cleared and mounted in benzyl alcohol–benzyl benzoate (Sive et al., 1998) in depression slides. Z-stacks of confocal images of fixed and cleared embryos were acquired using a Zeiss LSM 510 Axiovert using a C-Apochromat 10× 0.45 W objective lens with pinhole size of ca 2 Airy units at 0.7× scan zoom, which allows imaging the whole embryo. MR and fluorescence images were processed using Amira software (Mercury Computer Systems, Inc.). Animal cap tissue movement was measured using the measuring tool of the Amira software. For this, a two-color look-up table was applied to the images in a way that sharply distinguishes the animal and the vegetal tissue (see Fig. 1). The animal cap advancement between each five scans was measured at the lowest point of the animal cap-vegetal cell mass boundary as a linear distance. Contrast agent and fluorescent dye injection A polymeric gadolinium-based T1 contrast agent (P717 (Corot et al., 1997), provided by Guerbet Research, France) was supplied as an aqueous NaCl containing solution. To reduce the amount of sodium, P717 solution was desalted by diluting 10-times with reverse-osmosis-treated H2O and the volume then reduced to the original volume with a Microcon YM-10 centrifugal filter (Millipore, Billerica, MA). This procedure was carried out twice. Desalted P717 was supplemented with 25 mg/ml TexasRed Dextran 10 kDa (Molecular Probes, Oregon, USA). Injection capillaries were fabricated from quartz tubing O.D. 1 mm, I.D. 0.75 mm (Sutter Instrument Co, Novato, USA) with a Sutter P-2000 microelectrode puller. Contrast agents and fluorescent dyes were backfilled into the capillaries and intracellularly injected into individual blastomeres using a PLI-100 microinjector (Harvard Apparatus, Holliston, USA). Quantification of the injected amount was done by expelling dye into mineral oil on a haemocytometer before injections, then determining drop volumes. Single cell labeling was confirmed under a dissecting stereomicroscope equipped with fluorescence epi-illumination (Leica). Results Pregastrula epiboly moves animal cap tissue into the superficial dorsal marginal zone With the aid of intrinsic tissue specific contrast (Jacobs et al., 2003; Lee et al., 2007; Papan et al., 2007; Sehy et al., 2001), we can follow the morphogenetic movements of the animal cap and the vegetal cell mass in time series of developing embryos. Fig. 1 (see also supplementary Movie 1) shows a time-lapse sequence of pregastrula movements. At stage 8 (Fig. 1) the embryo is radially symmetric. No difference in tissue architecture between the dorsal and the ventral marginal zone is apparent, in agreement with histological observations (Hausen and Riebesell, 1991). A black line on the dorsal side and on the ventral side indicates the boundary between the bluelabeled animal cap tissue and the yellow-labeled vegetal cell mass. Using this line as a landmark, the vegetal-ward advancement of the animal cap can be observed in subsequent time-lapse frames (Fig. 1, stages 9–10.25). By stage 9, the dorsal side of the animal cap has just begun to advance vegetalward (white line in Fig. 1 stage 9). No advancement is observed on the ventral side. By stage 9.5, the dorsal side of the animal cap has further advanced but still no advancement is evident on 163 the ventral side. By stage 10, the animal cap has advanced about 220 μm on the dorsal side (white line in Fig. 1 stage 10), while no movement can be seen ventrally. The dorsal blastoporal lip becomes visible as a slight indentation (indicated by the black arrowhead). By stage 10.25, the animal cap has advanced about 290 μm from its original location, and now the ventral side too has begun to advance (ventral white line in Fig. 1 stage 10.25). The blastoporal lip is prominently visible (black arrowhead), but no invagination has occurred yet. For staging confirmation, the inset Figure at stage 10.25 shows an optical image of the embryo taken immediately after the MRI scan. The animal cap tissue moves into the superficial DMZ outside of the Cleft of Brachet (Brachet, 1935). Though the cleft is not directly visible in the MR images, intrinsic MRI tissue contrast and the well-known timing and location of its appearance (Hausen and Riebesell, 1991) allow defining the cleft between the mesendodermal and ectodermal germ layers, separating the superficial from the deep DMZ (white dashed line; stage 10.25). The asymmetry seen in the MR image of the stage 10.25 embryo correlates with the well-known histological appearance of a comparable stage embryo (Fig. 1 Hist; see also plate 18 in Hausen and Riebesell, 1991), where a domain of smaller cells is present in the DMZ, but not in the ventral marginal zone (VMZ). Timing of dorsal vs. ventral pregastrula epiboly The timing of the dorsal vs. the ventral pregastrula epiboly movement was found to be variable. In one embryo, the dorsal and the ventral side appeared to start moving at the same time and speed, while in three other embryos, the onset of the ventral movement lagged that of the dorsal movement by five, ten and twenty scans respectively. In two further embryos, both sides began moving at the same time, but the dorsal side moved faster compared to the ventral side. To quantify the difference of the pregastrula movement, we measured the advancement of the animal cap at the dorsal side and the ventral side in six embryos. The measurements are summarized in Fig. 2. The diagram shows the traveled Fig. 2. Pregastrula epiboly movement of the animal cap tissue, measured in six embryos. Abscissa: scan number (6:44 min/scan); Ordinate: distance in micrometers. Open circles: dorsal side of the animal cap; solid circles: ventral side of the animal cap. A 3rd order polynomial regression was used to fit the data. Dashed graph: dorsal; solid graph: ventral. 164 C. Papan et al. / Developmental Biology 305 (2007) 161–171 distance of the dorsal and ventral animal cap boundary as a function of the scan number. Image sequences were aligned with respect to the first sign of the upward movement of the mesendodermal mantle to provide a time frame independent of the animal cap movement. The polynomial regression lines indicate the average pregastrula epiboly movement of the dorsal and ventral side of the animal cap. It can be seen that, on average, the dorsal side begins to move about 10 scans (corresponding to 1 h 7 min) earlier compared to the ventral side. Time-lapse tracing of labeled blastomeres shows pregastrula apposition between the animal cap and the deep DMZ To define in more detail the spatial and temporal changes of the tissue relationships brought about by pregastrula movement, we traced the morphogenetic movements of B1- and C1blastomeres labeled at stage 6 with contrast agent through late blastula in time-lapse experiments. B1 descendants move into the superficial DMZ Fig. 3 (see also Supplementary Movie 2) shows selected images from a 2D (Figs. 3A–D) and a 3D time series (Figs. 3E– H) of two different embryos with a labeled B1-blastomere. Seven such embryos were analyzed. At stage 9 (Figs. 3A and E), the B1-clones are located mostly above the equator of the embryo. At stage 9.5 (Figs. 3B and F), the clones have begun to move vegetally; they continue to extend in the vegetal direction at stage 10 (Figs. 3D and G). By stage 10.25 (Figs. 3D and H), the clones have extended vegetally by about 200 μm and almost reach the blastoporal lip (indicated with white arrowheads in Figs. 3D and H). Both of the depicted clones are now located within the superficial DMZ outside of the Cleft of Brachet (indicated with a black dashed line in Figs. 3D and H). Thus, these time-lapse images show that pregastrula morphogenetic movement translocates cells from above the equator of the stage 9 embryo into the superficial DMZ of the stage 10 embryo prior to the onset of involution movements. Fig. 3. 2D (A–D) and 3D (E–H) time series showing how labeled B1-blastomere descendants move into the superficial dorsal marginal zone during late blastula stages. In panels E–H, the front part of the embryo is digitally cut away to facilitate visualization of internal structures. bc = blastocoel; veg = vegetal cell mass; asterisks = labeled cell clone; black dashed line: Cleft of Brachet. Embryonic stages (Nieuwkoop and Faber, 1994) are shown at the bottom of each panel. Arrowheads point to the dorsal blastoporal lip. Images are sagittal with dorsal to the right and the animal pole up. The medium surrounding the lower embryo is black because the Feridex added to the medium makes the water appear dark in MRI. Scale bar = 200 μm. C. Papan et al. / Developmental Biology 305 (2007) 161–171 C1 descendants move into the deep and superficial DMZ Fig. 4 (see also supplementary Movie 3) shows selected images from a 2D (Figs. 4A–D) and a 3D time series (Figs. 4E–H) of two different embryos with a labeled C1-blastomere. Nine C1-labeled embryos were analyzed. At stage 9 (Figs. 4A and E), the C1-clones are located at or slightly below the embryo's equator. The C1-clones span the entire thickness of the DMZ from the blastocoel floor to the embryo surface. By stage 9.5 (Figs. 4B and F), the superficial parts of the clones have moved vegetal-ward. This movement has become more apparent at stage 10 (Figs. 4C and G), when pregastrula epiboly has begun to move unlabeled tissue from the animal cap into the superficial DMZ (white arrows). In contrast, the deeper parts of the clones have not moved vegetal-ward to the 165 same extent; thus, a superficial layer of unlabeled tissue (white arrows in Fig. 4) comes to lie above the labeled deep tissue. By stage 10.25 (Figs. 4D and H), pregastrula epiboly movements have moved the outer part of the clones further vegetal-ward into the lower superficial DMZ; more unlabeled tissue has moved into the superficial DMZ superficial to the labeled deep DMZ. Thus, two distinct clone subsets can be distinguished: a large part in the deep DMZ inside the Cleft of Brachet (indicated with a black dashed line), and a second part in the lower superficial DMZ outside of the Cleft of Brachet. Tissue relationship examined by optical microscopy To confirm the observations made using MRI, we compared the stage 10 configurations of the B1- and the C1-clones using Fig. 4. 2D (A–D) and 3D (E–H) time series showing how labeled C1-blastomere descendants move into the deep and the superficial dorsal marginal zone during late blastula stages. Images are sagittal with dorsal to the right and the animal pole up. In panels E–H, the front part of the embryo is digitally cut away to facilitate visualization of internal structures. bc = blastocoel; veg = vegetal cell mass; asterisk = labeled cell clone; black dashed lines: Cleft of Brachet. The white dashed lines in panels E–H outline the blastocoel. White arrowheads point to the dorsal blastoporal lip. In 3D images, the blastoporal lip is not apparent, so the arrowhead indicates its approximate location. Embryonic stages (Nieuwkoop and Faber, 1994) are shown at the bottom of each panel. White arrows indicate the unlabeled animal cap tissue that moves into the superficial DMZ overlaying the labeled C1 descendants of the deep DMZ. Black arrowhead: C1-clone lining of the blastocoel floor; dotted white line: blastocoel floor lining of the VCM as defined by the intrinsic image contrast (see text). Scale bar = 200 μm. 166 C. Papan et al. / Developmental Biology 305 (2007) 161–171 confocal microscopy of embryos in which both blastomeres had been injected with different fluorophores at the 32-cell stage in the same embryo. While this does not allow the tracking of the developmental history of the labeled cells over time, it has the advantage of showing directly the complementary positions of the two neighboring labeled sets of clonal descendants at a particular time point in development. Fig. 5 shows two examples of stage 10 embryos in which the B1-blastomere was labeled with fluorescein and the C1blastomere with Texas red. The configuration of the dyelabeled descendants agrees with the observations made by the time-lapse sequences: the B1-clone is mostly located in the superficial marginal zone and the C1-clone makes a large contribution to the deep DMZ, underlying the B1-derived tissue in the superficial marginal zone. The C1-clone also contributes to the superficial DMZ just above the blastoporal lip. Note that the boundary between the clones roughly coincides with the Cleft of Brachet (indicated by dashed lines), which, due to the volume rendering technique, is not well defined in the images. In Fig. 5B, it is evident that cells of both clones straddle this boundary. For the case of the B1-clone, this means that most cells are located in the outer presumptive ectoderm, while some clonally related sibling cells are located within the mesendoderm. Interrelation of blastocoel floor expansion and DMZ formation Beginning at the onset of gastrulation, the blastocoel floor (BCF) expands (Winklbauer and Schürfeld, 1999), and cells labeled in the center of the blastocoel floor at stage 9 have been found to have moved toward the dorsal side of the embryo by stage 10.5 (Jones et al., 1999). We directly analyze the BCF expansion process from time-lapse sequences in further detail and relate it to the animal cap tissue as defined by labeled B1and C1-blastomeres. Timing of BCF expansion The timing of the blastocoel floor expansion can best be observed in a 2D time series as shown in Figs. 4A–D. The intrinsic contrast defines the edge of the blastocoel floor, which is indicated with a white dotted line. At stage 9 (Fig. 4A), the BCF is separated from unlabeled animal cap tissue by the labeled tissue of the C1-clone, which is exposed to the blastocoele (black arrowhead in Fig. 4A). As the blastocoel floor expands, the amount of labeled C1-tissue lining the blastocoel becomes successively reduced (Figs. 4B and C). At stage 10 (Fig. 4C), the clone retains only limited contact with the blastocoel floor (black arrowheads in Fig. 4C). By stage 10.25 (Fig. 4D), as the blastocoel floor has maximally expanded, the labeled cells have disappeared from the blastocoel lining because the BCF has overgrown the C1-clone. In this way, the BCF has moved into the upper deep marginal zone beneath the Cleft of Brachet, where it has now come into contact with unlabeled tissue from the animal cap. These images show clearly that the BCF expansion begins earlier than previously thought (at around stage 9) and cells of the BCF already contribute to the deep DMZ by the onset of gastrulation at stage 10. Note that the blastocoel floor overgrows the labeled clone cells because the whole BCF is elevating and thus raising above the original deep marginal zone (compare the level of the BCF in Figs. 4A–D and Supplemental Movie 3). However, at the same time the labeled clone cells destined to populate the deep marginal zone are moving downwards only little. The cells destined to form the outer marginal zone are moving down to a greater extend and in this way overlaying the deep marginal zone cells as explained above. This simultaneous movement can best be understood in the movie sequence of the C1-labeled embryo (Supplemental Movie 3). Site of the vegetal cell mass—animal cap apposition Fig. 5. 3D volume renderings of confocal image stacks of two stage 10 embryos showing the spatial relationship at the onset of gastrulation between a fluorescein-labeled B1-clone (rendered in green) and a Texas-red-labeled C1-clone (rendered in orange/red). Bc: blastocoel; Veg: vegetal cell mass; asterisk: site of the dorsal blastoporal lip; white arrowheads: deep portion of the C1-clone underlying the more superficial B1 cells (black arrowheads); white arrow: region of intermingling between the C1- and the B1-clones; dashed line: Cleft of Brachet. The Cleft of Brachet is not directly apparent in the images because the figures are volume renderings of an approximately 300-μm-thick image stack and the lower signal intensities of the unlabeled tissue set transparent. Scale bar = 200 μm. The site of the apposition between the rim of the vegetal cell mass (VCM) and the animal cap (AC) was determined from time-lapse sequences of embryos in which a B1-blastomere had been labeled. Fig. 6 shows a 2D time series (Figs. 6A and B) and a 3D time series (Figs. 6C and D) of two such labeled embryos. At stage 9 (Figs. 6A and C), the B1-clones are situated in the animal cap in both embryos. In the first embryo (Fig. 6A), the labeled clone cells are seen just above the embryo's equator, reaching 300 μm into the animal cap. In the second embryo (Fig. 6C), the labeled cells reach about 200 μm higher toward the animal pole. At stage 10 (Figs. 6B and D), pregastrula epiboly has shifted the clones into the DMZ and the blastocoel floor has expanded. In the first embryo, the clone has completely moved into the superficial DMZ and the edge of C. Papan et al. / Developmental Biology 305 (2007) 161–171 167 Fig. 6. Formation of the contact between the animal cap and the vegetal cell mass during late blastula. (A–B) 2D time series; (C–D) 3D time series with the front part of the embryo digitally cut away to facilitate visualization of internal features. Veg: vegetal cell mass; bc: blastocoel. A dashed line outlines the blastocoel in panels C and D. White arrowheads: blastoporal lip. Embryonic stages (Nieuwkoop and Faber, 1994) are shown in the upper left of each panel. In both embryos, a B1-blastomere was labeled with contrast agent at the 32-cell stage, but occupies different regions in the embryo due to variability in the early blastomere cleavage patterns. The clones are visible as a bright region in the animal cap at the dorsal side. The regions, which will contact each other, are indicated with an asterisk and the direction of movement is indicated with white arrows. At stage 9 (A, C), both clones are located in the lower animal cap. By stage 10 (B, D), morphogenetic movements have brought these tissue regions in close proximity to each other: In panel B, the upper limit of the clone is now on the level of the blastocoel floor, and in panel D, one half of the clone has moved into the superficial DMZ. At the same time, the blastocoel floor has expanded, now contacting the animal cap. Scale bar = 200 μm. the blastocoel floor is in apposition to the anterior/animal limit of the B1-clone (Fig. 6B). In the second embryo, only the posterior half of the labeled clone has moved into the DMZ, reflecting the initially more animal location of the clone, so that the edge of the blastocoel floor is contacting the middle of the clone (Fig. 6D). The contact site is indicated by asterisks. These images directly show that the edge of the blastocoel floor comes into contact with the animal cap by stage 10; by this stage, the vegetal cell mass has moved into the upper/anterior deep DMZ where it contacts the animal cap approximately at the boundary between the A-tier and the B-tier blastomeres. (DMZ) and Spemann organizer (Spemann and Mangold, 2001), could not be adequately described. Using time-lapse microscopic MRI, we were able to eliminate the problem of tissue opacity and observe the process of the DMZ formation noninvasively in live embryos. Because a single embryo is tracked over time, time-lapse MRI also overcomes the problem of embryo-to-embryo variability (Ewald et al., 2004; Masho, 1988), which makes the analysis of internal movements from time-course data difficult. From our studies, we found that pregastrula epiboly begins during late blastula around stage 9 and changes the structure of the dorsal marginal zone of the stage 10 embryo (Fig. 1). Discussion Pregastrula epiboly leads to marginal zone asymmetry Epiboly begins during late blastula Histological examinations have suggested that the first morphogenetic movements occur prior to stage 10 Xenopus gastrula embryos (Bauer et al., 1994; Brachet, 1935; Hausen and Riebesell, 1991; Holtfreter, 1943; Keller, 1978; Nieuwkoop and Florschütz, 1950; Schechtman, 1934; Vodicka and Gerhart, 1995; Vogt, 1929). However, due to the opacity of the amphibian embryos, the precise nature of these movements, and their role for the formation of the dorsal marginal zone From histological preparations, the tissue structure of the early Xenopus embryo is well known: at early-to-mid blastula stages, the embryo is rotationally symmetric, with smaller cells in the animal cap and larger cells in the vegetal cell mass. By stage 10, the internal structure the embryo has become asymmetric; the dorsal marginal zone is populated with smaller cells reaching deep into the embryo, while the ventral marginal zone is not (Hausen and Riebesell, 1991; Nieuwkoop and Florschütz, 1950). 168 C. Papan et al. / Developmental Biology 305 (2007) 161–171 Measurements of the pregastrula epiboly in six embryos (Fig. 2) showed some variability between the early pregastrula movements of the dorsal and ventral side of the embryo. In some cases, the dorsal side begun to move earlier, while in other cases both sides begun to move at the same time but faster on the dorsal side. In one case, both sides moved at the same time and speed. The average measurement from six embryos showed that pregastrula epiboly begins about 1:07 h earlier on the dorsal side than on the ventral side. From this, we suggest that the pregastrula epiboly movement establishes the dorso-ventral asymmetry of the marginal zone seen in histological images. Pregastrula epiboly movements resulting in dorso-ventral asymmetry of the embryo may be widespread among lower vertebrates, as a similar morphogenetic pattern is found in the zebrafish where the blastoderm advances from 30% to 50% epiboly prior to the onset of gastrulation (Kimmel et al., 1995). As a result, the rotationally symmetric blastula-stage zebrafish embryo becomes bilaterally symmetric well before the onset of the involution/ingression movements (Schmitz and CamposOrtega, 1994). Planar-to-vertical tissue rotation within the DMZ To investigate the tissue rearrangements in more detail, we studied the pregastrula morphogenetic movements in the DMZ with the aid of cell labeling (Figs. 3 and 4). At stage 9, the descendants on the B1- and the C1-blastomeres are in locations reminiscent of their locations at the time of labeling at stage 6. The B1-clone is located above or animal-ward of the C1-clone in a planar (also termed horizontal) juxtaposition. With the onset of the pregastrula epiboly movements, the B1-derived tissue moves into the DMZ outside of the Cleft of Brachet. Given that the C1 descendants in the deep DMZ remain in position, the downward-moving B1-tissue comes to overlie this C1-derived internal tissue. Thus, the tissue within the DMZ folds from a planar/horizontal into a radial/vertical juxtaposition, bringing non-involuting presumptive ectodermal tissue from the B-tier blastomere in vertical/radial contact with the deeper mesendodermal tissue of the C-tier blastomere before the onset of gastrulation and without involution. This shearing movement effectively generates an apposition similar to the result of an involution movement. An internal involution movement within the deep DMZ tissue characterizing the onset of gastrulation in Xenopus has been described previously by Nieuwkoop and Florschütz (1950). Based on their fixed histological sections the authors describe a rolling up of dorsal internal material around an internal lip at the beginning of gastrulation. Our study confirms the anatomy they report but shows that this apposition is not brought about by the movements they envisaged; instead, it results from the surface layer moving downward over the internal tissue. Therefore, this process should not be termed internal involution. Strong shifting of C1-derived tissue from a marginal/animal toward the dorso-vegetal position has been suggested previously. In a fate-mapping analysis for the Spemann organizer precursors (Bauer et al., 1994), the C1-blastomere was reported to rotate all the way from the equatorial region into the vegetal cell mass below the limit of the dorsal blastoporal lip. Our observations correct this notion by showing that there is no such strong displacement of the C1-blastomere (Vodicka and Gerhart, 1995). Spatial relation between neural and mesodermal precursors at the onset of gastrulation The time-lapse study of the B1-blastomere (Fig. 3) shows that tissue from this blastomere moves into the superficial DMZ and comes to overlie the deep tissue that originates from the C1blastomere. Fate mapping studies (Keller, 1975, 1976; Vodicka and Gerhart, 1995) show that the descendants of the B1blastomere at stage 10 (Fig. 3) contribute to the non-involuting dorsal marginal zone (NIDMZ), and thus will give rise to neuroectoderm. To clarify these relationships, Fig. 7 shows an overlay of an embryo in which a C1-blastomere was labeled with the surface fate map of the stage 10+ embryo (after Keller, 1975, modified). Our studies demonstrate that the stage 10+ presumptive neuroectoderm is underlain by C1-derived tissue, which previous studies have shown to possess neural-inducing capability (Jones et al., 1999; Lupo et al., 2002; Nieuwkoop and Koster, 1995). Thus, the pregastrula epiboly movement brings presumptive neuroectoderm into vertical contact with inductive mesendodermal precursors during late blastula, independent of the involution/invagination movements of gastrulation. A vertical relation between presumptive prechordal mesoderm and neuroectoderm in the Xenopus embryo at the onset of gastrulation was observed by (Nieuwkoop and Koster, 1995). Our study agrees with their original notion; however, the authors explain this relationship as occurring through an Fig. 7. Overlay of a sagittally oriented 3D NMR image of a stage 10.25 embryo with a stage 10+ surface fate map after Keller (modified) (Keller, 1975, 1976). The front half of the 3D NMR image has been digitally cut away to facilitate the visualization of the inner structures. The regions above the double line (white arrowhead) are presumptive ectoderm, and regions below the double line are presumptive mesendoderm. AN = anterior neuroectoderm; MN = midneuroectoderm; PN = posterior neuroectoderm; NIDMZ = non-involuting dorsal marginal zone; IDMZ = involuting dorsal marginal zone. The black dashed line indicates the Cleft of Brachet, which is forming at this developmental stage. White arrowhead = dorsal blastoporal lip. Scale bar: 200 μm. C. Papan et al. / Developmental Biology 305 (2007) 161–171 internal involution movement beginning at stage 10. Our data show that this interaction is brought about by simultaneous pregastrula epiboly and blastocoel floor expansion, beginning at stage 9. The role of blastocoel floor expansion in DMZ formation The onset of the blastocoel floor expansion (Winklbauer and Schürfeld, 1999) has been reported to begin at the time of the pigment line appearance at stage 10. Our direct time-lapse observations show that blastocoel floor expansion on the dorsal side is already beginning at stage 9, so that a considerable part of the apposition between the vegetal cell mass and the animal cap is already established by stage 10 (Figs. 4A–D). As the BCF expands, it overgrows the cells previously at the edge of the BCF (the labeled C1 descendants) coming to lay animal-ward of them. In this way, the anterior subset of the deep tissue of the DMZ (inside the Cleft of Brachet) is composed of descendants of the D-tier blastomeres, while its posterior subset is composed of C-tier descendants. It should be noted that the ventral side of the BCF does not significantly extend until the beginning of the ventral epiboly movement (data not shown). The site of apposition between the blastocoel floor and the animal cap at stage 10 is located approximately at the boundary between the A- and the B-tier descendants. The coincident motions of BCF expansion and epiboly move tissue vegetal mass and from the animal cap into the DMZ. This brings 169 presumptive anterior neuroectoderm into contact with the expanding margin of the blastocoel floor by stage 10. This is in agreement with observations by Koide et al. (2002), who showed that as early as stage 10.25, the leading edge of gastrulation has reached the anterior neuroectoderm in the animal cap. Model of DMZ formation in Xenopus laevis Based on time-lapse MRI data, we present a model for the pregastrula tissue movements leading to the formation of the DMZ of a stage 10 embryo (Fig. 8). At stage 9, morphogenetic movements begin to transform the dorsal marginal zone (Fig. 7A). Epiboly moves tissue from the animal cap vegetal-ward; the internal tissue from the C-region does not move to the same extent, as evident by the tilting of the interface between the Bregion (orange in Fig. 8) and the C-region (blue in Fig. 8). The shearing motion of the B-descendants over the C-descendants transforms the original animal–vegetal boundary between regions into an exterior–interior boundary, which approximately coincides with the Cleft of Brachet. At the same time, the blastocoel floor expands. Cells in a more medial location in the BCF overgrow the cells at the rim; by the onset of gastrulation at stage 10, they have come in direct contact with the animal cap cells. In this way, the deep DMZ underneath the non-involuting marginal zone is composed of cells from D-tier blastomeres (anterior) and from C-tier blastomeres (posterior). Fig. 8. Semi-schematic model depicting the morphogenetic movements and the resulting changes in tissue relationships during late blastula stages. The B-blastomere region is colored in orange and the C-blastomere region is colored in blue. Embryonic stages are shown below the figures. For details see text. Bc: blastocoel; Veg: vegetal cell mass. Arrowheads: dorsal blastoporal lip; IMZ: involuting marginal zone; NIMZ non-involuting marginal zone; dashed line: Cleft of Brachet. Scale bar = 200 μm. 170 C. Papan et al. / Developmental Biology 305 (2007) 161–171 Note that in our model, the boundary between the B- and the C-region coincides with the future Cleft of Brachet (dashed lines in Fig. 8). In embryos in which the B1-blastomere occupies a more vegetal position at stage 6 (due to the variability in early cleavage patterns; Masho, 1988, 1990; Wetts and Fraser, 1989), some of the B1 descendants would be found on the inside of the Cleft of Brachet (see Fig. 5B). This can explain why B-tier blastomeres, while giving rise to a large fraction of anterior neural tissue (retina, brain), often give rise to a significant populations of anterior mesodermal and endodermal tissue (Bauer et al., 1994; Dale and Slack, 1987). In an involution-based gastrulation model, this separation of fates would be problematic, as it must involve long-range cell migrations, which seem inconsistent with cell lineage studies (Wetts and Fraser, 1989). For example, the mesodermal descendants of the B1-blastomere would need to migrate toward the blastopore lip, involute and then move all the way to the animal pole (a distance of roughly 1 mm) in 6–8 h, while sibling cells with a neural fate would remain on the embryo's surface and move little. Our in vivo MRI observations offer a much simpler scenario: cleavages of the B1-blastomere separate it into deeper (anterior mesoderm) and superficial (neuroectoderm) daughters, which become separated by later shearing motions and mitoses. Because the B1 descendants fated to become anterior mesoderm are already in place in the deeper regions, there is no need for the long-range migrations required of the involution models. The rotation of the boundary between the B-descendants and the C-descendants from a horizontal/planar into a vertical/radial configuration resembles an “internal involution”. However, in a true involution movement, the internalizing tissue folds back over the external cell layer, distinct from the motions we document of the external tissue moving over the deeper tissue layers. Acknowledgments We thank Vladimir Korzh, Rachel S. Kraut and Rudolf Winklbauer for suggestions to improve the manuscript, Michael J. Tyszka and Seth Ruffins for technical discussion and Andrey Demyanenko for building the NMR coil. We thank Dr. Claire Corot (Guerbet Research, France) for the generous gift of the P717 contrast agent. This work was supported by the NIH Grant number HD25390. C.P. was in part supported by the Deutsche Forschungsgemeinschaft, Grant number PA 562/1-1. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2007.02.005. References Bauer, D.V., Huang, S., Moody, S.A., 1994. 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