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Development 127, 87-96 (2000) 87 Printed in Great Britain © The Company of Biologists Limited 2000 DEV3080

Formation of the avian from spatially restricted : evidence for polarized division in the elongating streak

Yan Wei and Takashi Mikawa* Department of Cell Biology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA *Author for correspondence (e-mail: [email protected])

Accepted 13 October; published on WWW 8 December 1999

SUMMARY

Gastrulation in the begins with the formation of a cells generated daughter cells that underwent a polarized primitive streak through which precursors of definitive cell division oriented perpendicular to the anteroposterior and ingress and migrate to their embryonic axis. The resulting daughter cell population was embryonic destinations. This organizing center for amniote arranged in a longitudinal array extending the complete is induced by signal(s) from the posterior length of the primitive streak. Furthermore, expression of margin of the blastodisc. The mode of action of these cVg1, a posterior margin-derived signal, at the anterior inductive signal(s) remains unresolved, since various marginal zone induced adjacent cells, but not those origins and developmental pathways of the primitive streak lateral to or distant from the signal, to form an ectopic have been proposed. In the present study, the fate of primitive streak. The cVg1-induced epiblast cells also chicken blastodermal cells was traced for the first time in exhibited polarized cell divisions during ectopic primitive ovo from prestreak stages XI-XII through HH stage 3, streak formation. These results suggest that blastoderm when the primitive streak is initially established and prior cells located immediately anterior to the posterior marginal to the migration of mesoderm. Using replication-defective zone, which secretes an inductive signal, undergo spatially retrovirus-mediated gene transfer and vital dye labeling, directed cytokineses during early primitive streak precursor cells of the stage 3 primitive streak were mapped formation. predominantly to a specific region where the embryonic midline crosses the posterior margin of the epiblast. No significant contribution to the early primitive streak was Key words: Primitive streak, Gastrulation, Fate map, Retrovirus, DiI, seen from the anterolateral epiblast. Instead, the precursor Chick, Epiblast, Cytokinesis

INTRODUCTION the epiblast by stage X (Eyal-Giladi and Kochav, 1976; Kochav et al., 1980). The underlying the epiblast begins to Gastrulation is the morphogenetic event through which the grow anteriorly from the posterior margin of the area pellucida three definitive germ layers, , mesoderm and at stage XI and covers the entire ventral surface of the area endoderm, are established. Coordinated behaviors of pellucida by stage XIII. Primitive streak formation is first embryonic cells during gastrulation have been carefully studied detected as a cluster of cells in the posterior margin of the area in non-, particularly frog (reviewed in Keller, pellucida along the embryonic midline at HH stage 2 1986). In the frog, animal hemisphere involute (Hamburger and Hamilton, 1951) and is completed by HH through the dorsal blastopore lip, undergo convergent- stage 3. Mesodermal cell migration begins by HH stage 4 as extension and move as a sheet on the inner surface of the Hensen’s node and groove are established. toward the animal pole of the . Embryos of Recent studies have identified several transcriptional higher amniotes, such as and , do not utilize factors and growth factor signals involved in primitive streak blastopore-mediated involution as the mechanism of formation and/or gastrulation (Stern et al., 1990; Knezevic et gastrulation. Instead, they initially develop a primitive streak al., 1995, 1997; Boncinelli and Mallamaci, 1995; Wilson et consisting of bottle-shaped mesenchymal cells along the al., 1995; Winnier et al., 1995; Seleiro et al., 1996). It has posterior half of the midline of the epithelioid epiblast. Once also been shown that epiblast cells of the area pellucida are the primitive streak is established, mesenchymal cells begin an competent to respond to inductive signaling and can be anterolateral migration to differentiate into embryonic induced to form an ectopic primitive streak (Khaner and Eyal- mesoderm and endoderm. Giladi, 1986; Stern, 1990; Mitrani and Shimoni, 1990; In the chicken embryo, blastoderm cells of the area pellucida Mitrani et al., 1990; Ziv et al., 1992; Callebaut and Van first differentiate into a single layer of epithelioid cells called Nueten, 1994; Bachvarova et al., 1998). The role(s) of these 88 Y. Wei and T. Mikawa factors in inducing the primitive streak is presently unclear, circle, site 3 was defined as the spot immediately inside the circle partly due to uncertainty in our understanding of the origin along a diagonal 135¡ from the apex of the AP axis, site 4 was the and development of the authentic primitive streak. The midpoint between sites 2 and 3, and site 5 was defined as the region pattern of primitive streak development might be critical for posterior to the midpoint of Koller’s sickle. Other general considering the mechanisms of inductive signaling. At least procedures for microsurgery, injection of virus and incubation of three distinct models have been proposed to describe infected embryos have been described previously (Mikawa et al., 1992a,b; Itoh et al., 1996). primitive streak formation, based on migration patterns of epiblast cells in cultured blastodiscs (Fig. 1). The first model Histology hypothesizes that epiblast cells migrate from lateral regions All embryos were fixed by injecting 2% paraformaldehyde in PBS towards the midline when they ingress to form the primitive underneath the embryonic discs. The fixed discs were then removed streak (reviewed in Balinsky, 1975). Another model presumes from the eggs and processed for morphological examination. Virally that HNK-1-positive cells, sparsely distributed within the tagged embryos were stained with X-gal for detection of β-gal in epiblast, individually ingress into the blastocoel, migrate to whole mount as described (Mikawa et al., 1992a). DiI in some the posterior midline and form the initial primitive streak labeled embryos was photoconverted, according to Ruiz i Altaba β (reviewed in Stern, 1992). The third model predicts that et al. (1993). Embryos exhibiting -gal-positive cells or posterolateral epiblast cells sequentially migrate to the photoconverted DiI precipitates were embedded in Epon or paraffin, serially sectioned at 4-7 µm thickness and examined by bright-field, midline and then move anteriorly along the midline (Eyal- phase or Hoffman Modulation Contrast optics. Actin and Giladi et al., 1992). chromosomes of stages XI, XII, 2+ and 3 were stained with In the present study, an in ovo protocol was established for fluorescein-conjugated phalloidin and DAPI (4′, 6-diamidino-2- tagging chicken blastoderm cells at prestreak stages (XI-XII) phnylindole, Molecular Probe), respectively. in order to trace primitive streak formation. Data are presented that show that the majority of precursor cells giving rise to the BrdU labeling HH stage 3 primitive streak are restricted to a midline area S-phase nuclei during primitive streak formation were labeled with within the posterior margin of the blastodisc. During early a thymidine analogue, BrdU (5′-bromo-2′deoxyuridine, Amersham). µ primitive streak formation, the precursor cells undergo At various times of incubation, 500 g of BrdU per egg were polarized cytokinesis oriented along the midline. Their introduced onto embryos through a small window in the egg shell and shell membrane. Eggs were reincubated for further daughter cells are then longitudinally arrayed along the development until stages 3− to 3. All embryos were then fixed primitive streak at HH stage 3. and removed from the eggs as above. After removal of unincorporated BrdU by washing with PBS, embryos were immunolabeled in whole mount with anti-BrdU antibodies MATERIALS AND METHODS (Amersham) followed by detection with the ABC kit (Vector lab) with a peroxidase-based substrate. BrdU-negative nuclei were Virus counterstained with DAPI (1 µg/ml). A replication-defective variant of spleen necrosis retrovirus (Dougherty and Temin, 1986) encoding bacterial β-galactosidase (β- Ectopic induction of primitive streak gal) was generated from a packaging cell line, D17.2G, as described Ectopic primitive streaks were induced by implanting COS cells (Mikawa et al., 1992a). The virus particles were concentrated to titers expressing a chick Vg1 (cVg1, gifts from Drs Claudio Stern and Jane of ~107-108 virions/ml (Mima et al., 1995) just before injection. Dodd, Columbia College of Physicians and Surgeons, NY), according Propagation of the β-gal viral vector in vitro and proof of helper to Shah et al. (1997). After 7-9 hours of incubation, the embryos were virus-free stocks have been presented elsewhere (Mikawa et al., fixed with 2% paraformaldehyde in PBS and those exhibiting an 1992b; Mikawa and Fischman, 1992). ectopic primitive streak were further processed for DAPI staining or in situ hybridization. In vivo labeling Fertilized chicken eggs were obtained from an outbred flock (Truslow In situ hybridization Farms, MD). A small window of ~20 mm diameter was opened in the Fixed embryos were hybridized with 1 µg/ml of DIG-labelled shell at the lateral side of the unincubated egg and the underlying shell antisense or sense RNA probe for chicken (provided by Dr membrane removed. Embryos were illuminated by cool light through Raymond B. Runyan, University of Arizona), according to Henrique a blue filter and monitored through a color videocamera (Hitachi et al. (1995) with the slight modification of substituting proteinase K VKC150) connected to a trinocular stereomicroscope. The monitored digestion with three 30 minute incubations with RIPA-buffer (50 mM image of embryos were examined to identify Koller’s sickle NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM characteristic of the posterior margin of stages XI-XIII prestreak EDTA, 50 mM Tris-HCl, pH 8.0). Immunodetection of the RNA embryos (Eyal-Giladi and Kochav, 1976). Embryos lacking an probes and color development were carried out according to Henrique obvious Koller’s sickle were not used. et al. (1995). DiI (1,1′-dioctadecyl 3,3,3′,3′-tetramethyl indocarbocyanine perchlorate: Molecular Probes, Inc.), at 0.25 % in dimethyl Photography and digital imaging formamide or concentrated viral suspensions containing 100 µg/ml All images of embryos and histological sections were photographed polybrene, were pressure injected in ovo (Mikawa and Gourdie, using Kodak Ektachrome 400 film or captured directly by a digital 1996) into one of five selected sites (Fig. 1) under the control of a camera, DKC-5000 (SONY, Japan). Color slides were scanned into Picospritzer II (General Valve Co., NJ). To map these sites within Adobe Photoshop 5.0 (Adobe Systems, Inc., Mountain View, CA) the blastodisc, the embryonic midline was first drawn on the monitor using a Nikon film scanner LS-1000 (Nikon, Japan). All images as the line intersecting the midpoint of Koller’s sickle. A complete were adjusted for brightness and contrast, and then cropped. To circle was then superimposed from the arc. Site 1 was demarcated normalize distribution of β-gal-positive cells, digital images from as the point where the midline meets Koller’s sickle, site 2 was six embryos were superimposed after each was adjusted to 16% designated as the region anterior to site 1 by 1/5 the diameter of the opacity. The origin of early primitive streak 89

RESULTS primitive streak in 75.1% of the tagged embryos (Fig. 3B). In the remainder of the embryos, β-gal+ cells were localized to Tagging of blastodiscs in ovo areas adjacent to the primitive streak, either anteriorly (3.9%), To identify the origin of the early primitive streak in ovo, five laterally (20.7%) or posteriorly (1.3%). Of the embryos sites of unincubated, prestreak-stage embryos were tagged and with primitive streak labeling, 35% exhibited β-gal+ cells the fate of labeled cells was examined at HH stage 3 prior to exclusively in the primitive streak (Fig. 3C), while 65% anterolateral migration of mesoderm (Fig. 1B). These sites contained β-gal+ cells both in the primitive streak and in were specifically selected to test the three models proposed for an area juxtaposed to the primitive streak. Histological the genesis of the primitive streak in the in vitro fate map examination of these embryos (Fig. 3C) confirmed that tagging studies (Fig. 1A). The predicted pattern of labeled cell of site 1 gave rise to the primitive streak which was richly distribution in each model is summarized in Fig. 1C. populated with β-gal+ cells. Tagging of other regions rarely In the previous fate map studies on cultured embryos, resulted in significant numbers of DiI+ or β-gal+ cells within Koller’s sickle representing the future posterior of the embryo the primitive streak (Fig. 4A-H). Instead, cells from site 2 has been a reliable landmark for detecting the embryonic (n=8) were usually found in the area anterior to the primitive anteroposterior (AP) axis, while the boundary between the area streak (Fig. 4A,E) and those from site 3 (n=22) were mainly pellucida and area opaca has been used to detect the embryonic distributed in the area lateral to the primitive streak (Fig. 4B,F). and extraembryonic boundaries. We first examined whether Marking of cells at site 4 (n=24) and site 5 (n=8) gave rise to these landmarks, which are clearly identifiable in vitro, were β-gal+ cells in the area anterolateral to the primitive streak detectable in ovo within the prestreak embryo. Fortunately, an (Fig. 4C,G) and in the extraembryonic region posterior to the arc- or crescent-shaped cluster of cells characteristic of primitive streak (Fig. 4D,H), respectively. Koller’s sickle was evident in ovo (Fig. 2A). To confirm that To normalize for variation in cell migration and targeting the arc-shaped cluster of cells is Koller’s sickle demarcating accuracy, the distribution patterns of tagged cells from 6 the posterior margin of epiblast, the midpoint of the arc was embryos were superimposed (Fig. 5). The data clearly labeled with DiI (Fig. 2A). Immediately after DiI labeling, demonstrated that in ovo the majority of cells forming the HH embryos were photographed (Fig. 2A), fixed and removed stage 3 primitive streak were derived from the posterior region from the egg (Fig. 2B). The DiI-labeled region was identified of the blastodisc, particularly from the midline portion (site 1). as the site where the embryonic AP axis intersected the Little contribution was evident from other areas surrounding midpoint of Koller’s sickle (Fig. 2B). Histological inspection site 1. Neither migration of precursor cells from the lateral of these embryos further proved that DiI was localized in epiblast and subsequent along the midline, nor Koller’s sickle, a cluster of cells protruding toward the ingression of precursor cells sparsely distributed in the epiblast underlying hypoblast (Fig. 2C,D). Sagittal sections of such and followed by accumulation to the posterior region was prestreak embryos revealed that the anterior portion of epiblast detected as the main route for generating the HH stage 3 was composed of a simple cuboidal , while the more primitive streak. These results suggest that the majority of posterior region was more stratified. This gradient in epithelial precursor cells giving rise to the HH stage 3 primitive streak aspect of the epiblast was used to verify the AP axis in the are already localized in the posterior-midline region of stages subsequent studies. Koller’s sickle was also used as a landmark XI-XII embryos. for tagging individual sites of prestreak blastodiscs in ovo. Developmental process of HH stage 3 primitive streak Migration pattern of labeled cells during primitive Although these results map the precursors of the HH stage 3 streak formation primitive streak predominantly to site 1 in the prestreak-stage The fate of tagged cells was examined at HH stage 3 (Fig. 3). embryo, it still does not explain how cells restricted to such a In the first set of experiments, blastoderm cells were tagged small area can generate the primitive streak, which expands to with DiI (Fig. 3A). DiI-positive (DiI+) cells from the posterior ~1 mm in length. Three potential hypotheses were tested (Fig. end of the midline (site 1) were predominantly localized to the 6). The first, based on the model proposed by Eyal-Giladi et primitive streak (Fig. 3A). The results indicated that DiI- al. (1992), assumes that cells localized to the midline region of labeled embryos developed normally and the migration pattern the precursor area first migrate cranially forming the most of the embryonic cells could be examined in ovo. The results anterior domain of the primitive streak, followed by laterally also suggested that cells of the HH stage 3 primitive streak localized precursors, which give rise to the more posterior predominantly arose from a small region within the posterior sections of the streak (Fig. 6A). The second hypothesis predicts margin of the embryonic midline. However, since the DiI that daughter cells from different regions of the precursor area signal fades after cycles of several cell division, there might randomly migrate along the midline and intermingled within have been a population of cells that lost DiI but had migrated the resulting primitive streak (Fig. 6B). The third hypothesis into the primitive streak. In addition, the DiI signal seen in HH assumes that individual cells within the precursor area migrate stage 3 embryos might have arisen from cells labeled at the and/or proliferate unidirectionally toward the anterior tip of the time of injection and from a second population that later passed primitive streak (Fig. 6C). through the DiI-deposit, since in several instances DiI crystals To test these possibilities, a subset of cells within the remained undissolved at the injection site. precursor region was labeled with β-gal and the distribution of To clarify those uncertainties of DiI-based cell tagging and β-gal+ cells in the resulting primitive streak was examined at tracing, cells of prestreak blastodiscs were genetically tagged HH stage 3. When a large number of precursor cells were with retrovirally encoded β-galactosidase (Fig. 3B). Marking tagged, there was no detectable pattern of β-gal+ cell of site 1 (n=82) gave rise to a β-galactosidase-positive (β-gal+) distribution in the primitive streak (Fig. 7A,B), as seen in the 90 Y. Wei and T. Mikawa

consistent only with the third model (Fig. 6C) in which individual precursor cells generate daughter cells arranged in a longitudinal array along the embryonic AP axis. Oriented cell division in the developing primitive streak This unique arrangement of daughter cells in a longitudinal array could be generated by elongation of individual cells and/or by oriented cell division along the midline. Cell shape and the orientation of cell division were both examined in the developing primitive streak and the non-gastrulating epiblast. Cell shapes were outlined by staining the actin-containing adherens belt with phalloidin, while the direction of cell division was predicted by the orientation of DAPI-stained metaphase chromosome plates. In the dorsal view of embryos stained with phalloidin, no elongation of cell shape along the AP axis was apparent in any area of the blastodisc. Instead, primitive streak cells were smaller (8.14±0.33 µm, n=96) than those in non-primitive streak epiblast layer (10.07±0.36 µm, Fig. 1. (A) Three distinct models proposed for genesis of the early n=96) (Fig. 9A,B). primitive streak. Blue lines indicate migration or movement of But an alignment of metaphase chromosome plates was primitive streak precursors. AP, anteroposterior axis. (B) Relative β clearly evident in the developing primitive streak, especially positions of five sites tagged with DiI or -gal virus at pre-streak when compared to other regions of the DAPI-stained embryos stages in vivo. (C) Predicted contribution of labeled cells (blue-filled circles) to the primitive streak by marking sites 1-5. (Fig. 9C,D). The majority of primitive streak cells in metaphase exhibited metaphase plates aligned perpendicular to the AP axis (Fig. 9C). This oriented plane of the metaphase above experiment (Fig. 3). However, when the retroviral titer chromosome plates in the streak was distinct from the random was reduced, a subdomain within the precursor area was orientation seen in non-primitive streak epiblast areas (Fig. tagged, giving rise to β-gal+ cells, which were arranged in a 9D). Quantitative data (Fig. 9E) showed that in the primitive longitudinal strand that often extended the entire length of the streak, about 50% of metaphase cells set up a plane of cell HH stage 3 primitive streak (Fig. 7C-F). Tagging of smaller division perpendicularly to the AP axis, and an additional 30% regions resulted in a narrower β-gal+ band. DiI-labeling gave of cells exhibited a plane tilted by more than 45¡ to the AP rise to similar results (not shown). Histological sections axis. Such polarized cell division was not evident once through embryos bearing a single β-gal+ stripe revealed that primitive streak elongation completed at stage 3+-4. These β-gal+ cells formed arrays in either the dorsal epithelioid or results suggest that, as the early primitive streak develops middle mesenchymal layers (Fig. 7G,H). Importantly, when a anteriorly along the midline, primitive streak cells undergo the few cells within the precursor area were tagged (Fig. 8A-C), oriented cytokinesis along the AP axis. β-gal+ daughter cells occupied only a portion of the entire The increase in cell number along the AP axis and the longitudinal array. consequence that this would have on the linear dimension of Such a unique arrangement of daughter cells was observed the early streak was established by counting the number of cells only when parental cells were tagged within the precursor area, and measuring their diameter (Table 1). Based on the results which extended anteriorly along the embryonic midline to 200- of DiI and viral tagging, the anterior boundary of the precursor 300 µm from the Koller’s sickle (Fig. 8C). These data are area extended 200-300 µm from the Koller’s sickle, while the

Fig. 2. (A) Dorsal view of a prestreak embryo immediately after DiI labeling (arrow) in ovo. Inset, higher magnification of DiI-labeled area. Note: DiI was introduced to the midpoint of the white arc (Koller’s sickle). (B) DiI- labeled embryos, fixed, isolated, photoconverted and viewed after removal of yolk. Inset: higher magnification of DiI-labeled (arrow) area. (C) Higher magnification of DiI-labeled area in D. (D) Montage of sagittal sections along the midline of DiI-labeled (arrow) prestreak embryo. a-p, anteroposterior axis; d-v, dorsal-ventral axis; e, epiblast; h, hypoblast, kc, Koller’s sickle; y, yolk. The origin of early primitive streak 91

primitive streak length of 1.27 mm was obtained by direct measurement of HH stage 3 embryos under a dissecting microscope. These values were then divided by the average diameter of precursor and primitive streak cells, 8.8 µm and 8.1 µm, respectively, giving rise to estimated cell numbers of 23-34 cells in the precursor area along the AP axis and 127 cells in the single column of the primitive streak. Thus, a 6- to 7-fold increase in cell numbers along the AP axis was estimated during primitive streak extension between prestreak- stage XII and HH stage 3 (Table 1). This estimate is consistent with the data obtained independently by counting cells of isolated, trypsinized fragments, in which 2.3×103 cells in the precursor area (n=4) and 1.4×104 cells in the early streak (n=3) were scored on average. These results suggest that precursor cells underwent three cell cycles on average during this 12 hour interval. This is consistent with the mitotic index (~2-4 hours, for cells of chicken gastrulae) which was independently obtained in earlier studies based on incorporation of a thymidine analogue (Sanders et al., 1993). However, since only a small number of cells were in the metaphase at the any given time point during primitive streak formation (Fig. 9C,D), it was uncertain whether primitive cells Fig. 3. Distribution of DiI+ (A) and β-gal+ (B,C) cells in HH stage 3 divided asynchronously and quickly completed M-phase or embryos which were marked at site 1. (A,B) Whole-mount dorsal only a subpopulation of primitive streak entered an active cell view; (C) a transverse section. ps, primitive streak; e, epiblast; h, cycle. These possibilities were tested by monitoring DNA hypoblast. synthesis of cells in developing primitive streak (Fig. 10). BrdU-positive nuclei were found throughout the primitive streak (Fig. 10A,B). Furthermore, the majority of primitive streak cells were labeled with BrdU within a few hours of incubation (Fig. 10C), indicating that the majority of cells in the developing primitive streak undergo an active, asynchronized cell cycle. Vg1 induce adjacent epiblast cells to form an ectopic primitive streak These results show that epiblast

Fig. 4. Distribution of DiI+ (A-D) and β-gal+ (E-H) cells in HH stage 3 embryos which were marked at site 2 (A,E), site 3 (B,F), site 4 (C,G) and site 5 (D,H).

Fig. 5. Probability in distribution of daughter cells derived from the tagged sites of prestreak embryos at HH stage 3. Digital images of six embryos from each labeling group were superimposed. Each number corresponds to the tagged site indicated in the diagram of prestreak embryos. Bar cord grades the presence of tagged cells in individual area; deeper blue represents the highest probability. 92 Y. Wei and T. Mikawa

Table 1. Primitive streak extension between stages XII and 3 Factor Score Length along the AP axis (l) Primitive streak (lps) 1,270±84 µm (n=8) Precursor area (lpa) 200 µmϹlpaϹ300 µm (n=18)

Cell diameter (¯) Primitive streak cells (¯ps) 8.14±0.33 µm (n=96) Precursor area cells (¯pa) 8.8±0.36 µm (n=92)

Estimated cell numbers along the AP axis (l/¯) Primitive streak (l/¯)ps (l/¯)ps=123.7±29.0 Precursor area (l/¯)pa 22.7Ϲ(l/¯)paϹ34.1

The maximal ps/pa ratio in Fig. 6. Three distinct models proposed for the early primitive streak Length = lps/minlpa 6.35 formation. See text. Cell number = (l/¯)ps/min (l/¯)pa 5.45

Required cell cycle (log2ps/pa) in increase of cells immediately anterior to the posterior marginal zone are Length = log2lps/minlpa 2.67 Cell number = log (l/¯) /min (l/¯) 2.45 the main precursors of stage 3 primitive streak. Therefore, it is 2 ps pa likely that a posterior marginal zone-derived signal induces (lps) of HH stage 3 embryos was directly measured under a dissect primitive streak by recruiting adjacent epiblast cells, rather microscope and presented as a mean value with 95% confidence interval, than attracting either a lateral or anterior population to migrate while (lpa) was estimated based on the results of Fig. 8D. (¯) was obtained by towards the midline. To test this possibility, COS cells analysing Phalloidin-stained embryos as in Fig. 9. expressing cVg1 (cVg1/COS), one of the primitive-streak- inducing factors, were placed at the marginal zone in the hours after implantation, a primitive-streak-like cluster of cells anterior portion of prestreak-stage embryos. Within 12-14 formed at the site where cVg1/COS was implanted (Fig.

Fig. 7. Development of the early primitive streak by unidirectional growth of precursors along the AP axis. The precursor region was labeled with β-gal virus either broadly (A,B) or discretely (C-F). Distribution of β-gal+ cell arrays (arrows) within the primitive streak was examined by transverse histological sections of discretely labeled embryos (G,H). Diagrams illustrate relative positions of labeled cells within the primitive streak.

Fig. 8. β-gal+ arrays oriented along the AP axis (A-C). A few cells in the precursor region were labeled with β-gal virus. (D) Frequency of primitive streak labeling (n=16) depends on distance (d in left diagram) of a tagging site from Koller’s sickle along the embryonic midline (arrow). Bar graph represents % of embryos exhibiting tagged cells only in primitive streak (filled bar), in both primitive streak and anterior epiblast (shaded bar), or only in anterior epiblast (open bar). The origin of early primitive streak 93

Fig. 9. Oriented metaphase chromosome plates in the early primitive streak. HH stages 3− embryos were stained with fluorescent phalloidin for actin (A,B) and with DAPI for nuclei (C,D). The developing primitive streak (A,C) and non-primitive streak epiblast (B,D) were examined. (E) Angle of metaphase chromosome plates to the AP axis (AP) were scored in the primitive streak (filled bar) and non-primitive streak epiblast (shaded bar). Primitive streak nuclei were examined in both the upper epithelioid layer and the middle mesenchymal layer. n: total number of counted metaphase nuclei in the primitive streak (ps) and non-primitive streak epiblast (epi).

11A,B) as shown by Shah et al. (1997). In situ hybridization similarly to those seen in authentic primitive streaks (Fig. 9). of a primitive streak marker, Brachyury, identified that the The results indicate that only epiblast cells adjacent to an cVg1-induced cluster of cells was indeed an ectopic primitive inductive signal undergo the primitive streak formation and streak (Fig. 11C). To determine the source of cells forming these exhibit a polarized cell division. cVg1-induced, ectopic primitive streak, epiblast cells at various positions relative to cVg1/COS were labeled with DiI and DiO (Fig. 11D-G). Marking of cells lateral or distant from DISCUSSION cVg1/COS gave rise to an unlabeled primitive streak (Fig. 11E). By contrast, labeling of epiblast cells immediately Using both DiI and retroviral cell tagging procedures, the adjacent to cVg1/COS frequently generated a DiI+ or DiO+ present study visualizes for the first time the in ovo primitive streak (Fig. 11F,G). The plane of cell division was development pattern of chicken blastoderm cells during then examined in cVg1-induced primitive streak. Metaphase formation of the early primitive streak. The data map early chromosome plates in cVg1-induced primitive streak were primitive streak precursors to a small area where the midline aligned perpendicularly to the ectopic streak axis (Fig. 11H), crosses the posterior margin (Koller’s sickle) of the prestreak blastodisc. Vg1-induced primitive streak formation strongly suggests that individual blastoderm cells adjacent to an inductive signal proliferate along the AP axis and exhibit polarized cell division during HH stage 3 primitive streak formation. This study examined the fate of cells from five selected sites of prestreak embryos. DiI and retroviral tagging only of site 1 produced labeled cells throughout the primitive streak. A DiI+ or β-gal+ primitive streak was never generated by labelling the other four sites. Our results suggest that neither epiblast cell migration from lateral to the midline nor random ingression of epiblast cells followed by subsequent accumulation to the posterior region is the primary pathway for generating the HH stage 3 primitive streak in ovo. Rather, these results define a model in which primitive streak precursor cells are already localized to the posterior-midline region of stages XI-XII blastodiscs. This conclusion is further supported by our present results that exogenous Vg1 induces an ectopic primitive streak Fig. 10. BrdU incorporation during primitive streak formation. by recruiting cells adjacent to the signal but not those lateral (A) Dorsal view of HH stage 3 primitive streak incubated with BrdU or distal from it. These results also corroborate previous for 2 hours before fixation, stained with BrdU antibody (left panel) and DAPI (right panel). Dark-brown (left panel) and blue (right observations that the epiblast portion of the posterior marginal panel) spots are BrdU-positive nuclei, while white spots (right panel) zone contributes to the primitive streak (Stern, 1990; Eyal- are BrdU-negative nuclei. (B) Time course of BrdU incorporation Giladi et al., 1992; Bachvarova, 1998). during primitive streak formation. The ratio of BrdU-positive nuclei These in ovo results differ from previously proposed models to total nuclei counted (n) is presented with standard deviation (bar). for the location and migration pathways of precursors 94 Y. Wei and T. Mikawa

Fig. 11. Induced primitive streak exhibits same characteristics as authentic primitive streak. Dorsal views of embryos into which cVg1-negative (A) and cVg1-positive (B,C) COS cells (arrows) were implanted. Authentic (ps) and induced (ips) primitive streaks were identified by in situ hybridization for Brachyury (C). The origin of Vg1-induced primitive streak was determined by labeling epiblast cells with DiI (e1) and DiA (e2 and g) (D). The anteroposterior polarity was illustrated by a and p. The localization of tagged cells from each site was indicated in fluorescent images (E,G) of resulting embryos. (F) Bright-field image of G. Implanted COS cells are indicated by white circles, while authentic (ps) and induced (ips) primitive streaks were outlined by dotted lines. (H) Angle of metaphase chromosome plates were scored in Vg1-induced streak as in Fig. 9. generating the early primitive streak (reviewed in Stern, 1992; Eyal-Giladi et al., 1992). Although the reason for this variation is not fully understood, significantly different experimental approaches were used to analyze cell movement in these studies. Model-b (Fig. 1A) is based on the expression pattern of HNK-1, a sulfated form of glucuronic acid, and the fate of an internalized HNK1-antibody complex (Stern, 1992). Model-c (Fig. 1A) is from the lateral epiblast. For example, at HH stage 4, cells that based on the fate of a tissue fragment implanted into an isolated originally formed the HH stage 3 primitive streak migrate blastodisc (Eyal-Giladi et al., 1992) and on the distribution of laterally and anteriorly and some of them differentiate into diffusible vital dyes in ovo (Gräper, 1929). By contrast, the lateral mesoderm (Eyal-Giladi et al., 1992; Garcia-Martinez present study directly traced the fate of blastoderm cells using and Schoenwolf, 1993), while the HH stage 4 primitive streak two non-invasive, non-diffusible cell tagging methods, DiI consists of cells that were originally localized in the labeling and retroviral-mediated gene transfer. Importantly, posterolateral region of the epiblast (Stern et al., 1995; Hatada these two independent in ovo approaches led to the same and Stern, 1994). Thus, the origin and migration pattern of cells conclusion. generating the early primitive streak are different from those The posterior-midline epiblast, including the primitive constituting the later stage primitive streak. precursor area, is stratified and not a simple epithelium. Our Interestingly, the β-gal-labelled cells were arrayed injection of higher viral titer into the precursor area, probably longitudinally in the early primitive streak. Potential tagging both the surface and deeper layer of epiblast cells, mechanisms by which progenitor cells at the posterior margin results in β-gal+ cells distributed to both the surface and deeper give rise to the extended streak include directed cell layer of the primitive streak (Fig. 3C). In contrast, injection of intercalation, directed cell migration, cell shape change and lower viral titers to the area tagged a population of cells in the oriented cell division. Mitotic activity has been shown to be surface layer (Fig. 7G) or in the middle layer (Fig. 7H). associated with primitive streak development (Sanders et al., Consistent with our observations, it has been shown that cells 1993), and β-gal expression in cells forming the early primitive of both the surface and deeper layer of the posterior-midline streak requires at least one cell cycle for integration and epiblast generate a subpopulation of primitive streak cells expression. This is clearly different from the non-mitotic (Bachvarova et al., 1998). The origin of these posterior-midline convergent and extension mechanism underlying frog cells of the stage XI-XII embryos remains to be identified. gastrulation (reviewed in Keller, 1986). Furthermore, cells of This study examined the location and developmental pattern the developing avian primitive streak do not exhibit elongation of precursor cells that form the HH stage 3 primitive streak. At perpendicular to the AP axis, the cell shape change predicted later stages, the primitive streak is composed of cells derived to occur with lateral-to-medial cell sliding during

Fig. 12. Schematic illustration of the directed cell division underlying the early primitive streak development along the AP axis. Blue spot in the left diagram marks the primitive streak precursor area at prestreak stages (X- XII), while blue bar in the right diagram represents HH stage 3 primitive streak (ps). In between, polarized cell division of precursor cells are illustrated. Blue arrows, inductive signal; AP, anteroposterior axis. The origin of early primitive streak 95 convergent/extension (Keller, 1986). Our results do not support Duband, J. L. and Thiery, J. P. (1982). Appearance and distribution of oriented cell intercalation as a major mechanism accounting fibronectin during chick embryo gastrulation and . Dev. Biol. 94, for primitive streak development. Indeed, β-gal+ cells are 337-350. Eyal-Giladi, H. and Kochav, S. (1976). From to primitive streak arranged in a longitudinal array without significant interruption formation: a complementary normal table and a new look at the first stages by β-gal-negative cells. A number of studies have of the development of the chick (I). Dev. Biol. 49, 321-337. demonstrated that migration of mesodermal cells is directed by Eyal-Giladi, H., Debby, A. and Harel, N. (1992). The posterior section of a fibronectin cue (Harrison et al., 1993). However, fibronectin the chick’s area pellucida and its involvement in hypoblast and primitive streak formation. Development 116, 819-830. is absent from the primitive streak (Duband and Thiery, 1982). Garcia-Martinez, V. and Schoenwolf, G. C. (1993). Primitive-streak origin The hypothesis that the hypoblast underlying the epiblast may of the cardiovascular system in avian embryos. Dev. Biol. 159, 706-719. provide a cue for direction of primitive streak development is Gräper, L. (1929). Die primitiventwicklung des huhnchens nach controversial (Azar and Eyal-Giladi, 1981; Khaner, 1995). stereokinematographischen untersuchungen, kontrolliert durch vitale Polarized cell division plays a role in shaping the neural farbmarkierung und verglichen mit der entwicklung anderer wirbeltiere. W. Roux Arch. EntwMech Org. 116, 382-429. plate (Hamburger, 1948; Martin, 1967; Sausedo et al., 1997; Hamberger, V. (1948). The mitotic patterns in the spinal cord of the chick Concha and Adams, 1998). Here we demonstrate that cells in embryo and their relation to histogenetic processes. J. Comp. Neurol. 88, the developing primitive streak have metaphase chromosome 221-284. plates with a plane perpendicular to the AP axis. Cells in an Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morph. 88, 49-67. ectopic primitive streak induced by Vg1 exhibit similar Harrisson, F., Nassauw, L. V., Hoof, J. V. and Foidart, J. M. (1993). polarity in their cell division. The orientation of metaphase Microinjection of antifibronectin antibodies in the chicken blastoderm: chromosome plates together with the observed longitudinal inhibition of mesoblast cell migration but not of cell ingression at the arrays of β-gal+ cells in the primitive streak suggest that primitive streak. Anat. Rec. 236, 685-696. directional cell division plays a role in primitive streak Hatada, Y. and Stern, C. D. (1994). A fate map of the epiblast of the early chick embryo. Development 120, 2879-2889. elongation along the midline. This idea is consistent with the Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, model illustrated in Fig. 12, where a local signal(s), such as D. (1995). Expression of Delta homologue in prospective neurons in the epithelial scatter factor (Stern et al., 1990), activin (Mitrani et chick. Nature 375, 787-790. al., 1990; Ziv et al., 1992) and Vg1 (Seleiro et al., 1996), in a Itoh, N., Mima, T. and Mikawa, T. (1996). Loss of fibroblast growth factor receptors is necessary for terminal differentiation of embryonic limb muscle. confined area of the posterior marginal zone, induces a Development 122, 291-300. blastoderm cell subpopulation that differentiates into the early Keller, R. E. (1986). The cellular basis of amphibian gastrulation. In primitive streak. During this process, primitive streak cells : A Comprehensive Synthesis (ed. L. Browder), Vol. undergo cytokinesis with the plane perpendicular to the AP 2, pp. 241-327. New York: Plenum Press. axis. Future studies should address mechanisms that orient the Khaner, O. (1995). The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast. Proc. Natl. Acad. Sci. USA 92, 10733- plane of cytokinesis in streak-forming cells. It remains to be 10737. established if oriented cell division is causal for primitive Khaner, O. and Eyal-Giladi, H. (1986). The embryo-forming potency of the streak elongation or an associated phenomenon. posterior marginal zone in stages X through XII of the chick. Dev. Biol. 115, 275-281. The authors thank Drs I. Skromne, C. D. Stern and J. Dodd for Knezevic, V., Ranson, M. and Mackem, S. (1995). The organizer-associated cVg1 cDNA and their guidance of ectopic primitive streak induction chick homeobox gene, Gnot1, is expressed before gastrulation and regulated synergistically by activin and retinoic acid. Dev. Biol. 171, 458-470. with Vg1. Our thanks extend to Drs P. Wilson, D. A. Fischman, J. Knezevic, V., DeSanto, R. and Mackem, S. (1997). Two novel chick T-box Hyer and K. Kelly for their comments and discussions on this study genes related to mouse Brachyury are expressed in different, non- and Ms L. Cohen-Gould, L. Miroff and L.-L. Ong for their technical overlapping mesodermal domains during gastrulation. Development 124, assistance. This work was supported in part by grants from the NIH 411-419. and the Mathers’ Foundation. T. M. is an Irma T. Hirschl Scholar. Kochav, S., Ginsburg, M. and Eyal-Giladi, H. (1980). From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick (II). Dev. Biol. 79, 296- REFERENCES 308. Martin, A. H. (1967). Significance of mitotic spindle fibre orientation in the Azar, Y. and Eyal-Giladi, H. (1981). Interaction of epiblast and hypoblast in neural tube. Nature 216, 1133-1134 the formation of the primitive streak and the embryonic axis in chick, as Mikawa, T., Borisov, A., Brown, A. M. C. and Fischman, D. A. (1992a). revealed by hypoblast-rotation experiments. J. Embryol. Exp. Morph 61, Clonal analysis of cardiac morphogenesis in the chicken embryo using a 133-144. replication-defective retrovirus: I. formation of the ventricular myocardium. Bachvarova, R. F., Skromne, I. and Stern, C. D. (1998). Induction of Dev. Dyn. 193, 11-23. primitive streak and Hensen’s node by the posterior marginal zone in the Mikawa, T., Cohen-Gould, L. and Fischman, D. A. (1992b). Clonal analysis early chick embryo. Development 125, 3521-3534. of cardiac morphogenesis in the chicken embryo using a replication- Balinsky, B. I. (1975). Introduction to . Philadelphia: Saunders. defective retrovirusIII: Polyclonal origin of adjacent ventricular myovytes. Boncinelli, E. and Mallamaci, A. (1995). Homeobox genes in vertebrate Dev. Dyn. 195, 133-141. gastrulation. Curr. Opin. Genet. Dev. 5, 619-627. Mikawa, T. and Fischman, D. A. (1992). Retroviral analysis of cardiac Callebaut, M. and Van Nueten, E. (1994). Rauber’s (Koller’s) Sickle: The morphogenesis: discontinuous formation of coronary vessels. Proc. Natl. early gastrulation organizer of the avian blastoderm. Eur. J. Morph. 32, 35- Acad. Sci USA 89, 9504-9508. 48. Mikawa, T. and Gourdie, R. G. (1996). Pericardial mesoderm generates a Cheng, N. N., Kirby, C. M. and Kemphues, K. J. (1995). Control of cleavage population of coronary smooth muscle cells migrating into the heart along spindle orientation in C. elegans: the role of the genes par-2 and par-3. with ingrowth of the epicardial organ. Dev. Biol. 173, 221-232. Genetics 139, 549-559. Mima, T., Ueno, H., Fischman, D. A., Williams, L. T. and Mikawa, T. Concha, M. L. and Adams, R. J. (1998). Oriented cell divisions and cellular (1995). FGF-receptor is reguired for in vivo cardiac myocyte proliferation morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis. at early embryonic stages of heart development. Proc. Natl. Acad. Sci. USA Development 125, 983-994. 92, 467-471. Dougherty, J. P. and Temin, H. M. (1986). High mutation rate of a spleen Mitrani, E. and Shimoni, Y. (1990). Induction by soluble factors of organized necrosis virus-based retrovirus vector. Mol. Cell. Biol. 168, 4387-4395. axial structures in chick . Science 247, 1092-1094. 96 Y. Wei and T. Mikawa

Mitrani, E., Ziv, T., Thomsen, G., Shimoni, Y., Melton, D. A. and Bril, A. Stern, C. D. (1990). The marginal zone and its contribution to the hypoblast (1990). Activin can induce the formation of axial structures and is expressed and primitive streak of the chick embryo. Development 109, 667-682. in the hypoblast of the chick. Cell 63, 495-501. Stern, C. D. (1992). Mesoderm induction and development of the embryonic Ruiz i Altaba, A., Warga, R. M. and Stern, C. D. (1993). Fate maps and cell axis in amniotes. Trends Genet. 8, 158-163. lineage analysis. In Essential Developmental Biology (ed. D. D. Stern and Stern, C. D., Ireland, G. W., Herrick, S. E., Gherardi, E., Gray, J., P. W. H. Holland). pp81-95. Oxford: IRL Press. Perryman, M. and Stoker, M. (1990). Epithelial scatter factor and Sanders, E. J., Varedi, M. and French, A. S. (1993). Cell proliferation in the development of the chick embryonic axis. Development 110,1271-1284. gastrulating chick embryos: a study using BrdU incorporation and PCNA Stern, C. D., Yu, R. T., Kakizuka, A., Kintner, C. R., Mathews, L. S., Vale, localization. Development 118, 389-399. W. W., Evans, R. M. and Umesono, K. (1995). Activin and its receptors Sausedo, R. A., Smith, J. L. and Schoenwolf, G. C. (1997). Role of during gastrulation and the later phases of mesoderm development in the nonrandomly oriented cell division in shaping and bending of the neural chick embryo. Dev. Biol. 172, 192-205. plate. J. Comp. Neurol. 381, 473-488. Wilson, V., Manson, L., Skarnes, W. C. And Beddington, R. S. (1995). The Seleiro, A. P. E., Connolly, D. J. and Cooke, J. (1996). Early developmental T gene is necessary for normal mesodermal morphogenetic cell movements expression and experimental axis determination by the chicken Vg1 gene. during gastrulation. Development 121, 877-886. Curr. Biol. 6, 1476-1486. Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. M. (1995). Bone Shah, S. B., Skromne, I., Hume, C. R., Kessier, D. S., Lee, K. J., Stern, Morphogenetic protein-4 is required for mesoderm formation and patterning C. D. and Dodd, J. (1997). Misexpression of chick Vg1 in the in the mouse. Genes Dev. 9, 2105-2116. marginal zone induces primitive streak formation. Development 124, Ziv, T., Shimoni, Y. and Mitrani, E. (1992). Activin can generate ectopic 5127-5138. axial structures in chick blastoderm explants. Development 115, 689-694.