The Spemann organizer meets the anterior-most neuroectoderm at the equator of early gastrulae in amphibian species

Abstract

The dorsal blastopore lip (known as the Spemann organizer) is important for making the body plan in amphibian gastrulation. The organizer is believed to involute inward and migrate animally to make physical contact with the prospective head neuroectoderm at the blastocoel roof of mid- to late-gastrula. However, we found that this physical contact was already established at the equatorial region of very early gastrula in a wide variety of amphibian species. Here we propose a unified model of amphibian gastrulation movement. In the model, the organizer is present at the blastocoel roof of blastulae, moves vegetally to locate at the region that lies from the blastocoel floor to the dorsal lip at the onset of gastrulation. The organizer located at the blastocoel floor contributes to the anterior axial mesoderm including the prechordal plate, and the organizer at the dorsal lip ends up as the posterior axial mesoderm. During the early step of gastrulation, the anterior organizer moves to establish the physical contact with the prospective neuroectoderm through the "subduction and zippering" movements. Subduction makes a trench between the anterior organizer and the prospective neuroectoderm, and the tissues face each other via the trench. Zippering movement, with forming Brachet's cleft, gradually closes the gap to establish the contact between them. The contact is completed at the equator of early gastrulae and it continues throughout the gastrulation. After the contact is established, the dorsal axis is formed posteriorly, but not anteriorly. The model also implies the possibility of constructing a common model of gastrulation among chordate species.

Keywords: Spemann organizer; amphibian; chordate; gastrulation; movement.

Figure 10

Conventional model and Proposed model of amphibian gastrulation. (A) Conventional mode of amphibian gastrulation. (B) Proposed model of amphibian gastrulation “S&Z movement”. The organizer and the prospective neuroectoderm are represented by red and blue, respectively. (A-1) Blastula embryo. (A-2, 3, 4, 5) The organizer which is derived from the dorsal marginal zone, invaginates, involutes into the body, and then migrates toward the animal pole on the inner surface of the blastocoel roof. (A-6) Anterior contact establishment (ACE) occurs around the animal pole area of mid- to late-gastrula embryo. (B-1) Blastula embryo. (B-2,3) The embryos undergo subduction movement. (B-4, 5) The zippering movement leads to physical contact between the blastocoel floor and the blastocoel roof. ACE occurs at equatorial region of very early gastrula. (B-6) The leading edge tissue migrates animally beyond the region of anterior contact. (B-7) An axial structure is progressively formed toward the posterior during the rest of the gastrulation movement. Curved arrows between A and P indicate the direction of axial structure formation. , organizer; , prospective neuroectoderm.

Figure 9

Model of “subduction and zippering” movement. The organizer and the prospective neuroectoderm are represented by red and blue, respectively, and the future A-P axis is indicated by numbers. The grey broken line indicates the blastocoel floor (equatorial) level of the embryo. (A) The organizer and the prospective neuroectoderm are aligned in tandem in the dorsal blastocoel roof at the blastula stage. (B–D) The “subduction” movement. (B) The prospective neuroectoderm (blastocoel roof) and the anterior organizer (blastocoel floor) are at right angles to each other at the onset of gastrulation. (C) The blastocoel floor is pushed down by the epiboly-like movement of the blastocoel roof (arrows). (D) The anterior organizer is dragged down, and the movements lead to the formation of a trench with the prospective neuroectoderm. (D–F) The “zippering” movement. (D) The anterior organizer and the prospective neuroectoderm face each other with the trench in between. (E) The red side of the trench approaches the blue side by vegetal rotation-like movement (curved arrow). (F) Then, the anterior organizer and the prospective neuroectoderm make physical contact, and Brachet's cleft (white broken line) is formed. The conceptual diagram of the “subduction and zippering” movement is shown below. Note that if the arrangement of the cells does not change in the region, the organizer seems to turn around during gastrulation (compare panel [A] with panel [F]). The dorsal lip is known to contribute to the pharyngeal endoderm (Vogt ; Nakamura ; Keller ; and see below). , organizer; , prospective neuroectoderm.

Figure 1

Comparison of timing when leading edge tissue covers inner surface of entire prospective neuroectoderm among amphibian species. (A–C) Schematic representation of neutral red labeling experiment. Blastocoels in embryos at the gastrula stage were injected with neutral red. Once the blastocoel roof contacted the inner tissue directly, the area of contact would not be stained by neutral red. When they grow up to neurulae, the stained state of the neural plate would enable us to know how the inner tissue lined the neuroectoderm at the injection time. The stippled area represents the prospective neuroectoderm (top panel) or the neural plate (middle panel). Bottom panels show Hynobius nebulosus neurulae whose blastocoels at the gastrula stage were injected with neutral red. The neural plate was entirely stained (type-A, A); the anterior portion of the neural plate was stained (type-B, B); no staining of the neural plate took place (type-C, C). Note that type-C includes embryos whose neural crests were labeled without neural plate staining. Epi, epidermis; NR, neutral red, NP, neural plate. (D) Graph of percentages of type-C neurula embryos in various amphibian species. In the horizontal axis, the injection time is expressed as the ratio of the length of time of blastopore appearance (“0”) to that of circular blastopore pigment line formation (“1”). Graph legends are shown on the right side. The number of embryos examined each time is shown in Table1. , Bombina orientalis; , Rana japonica; , Rana porosa brevipoda; , Rana rugosa; , Silurana tropicalis; , Xenopus laevis; , Ambystoma mexicanum; , Cynops ensicauda; , Cynops pyrrhogaster; , Hynobius nebulosus.

Figure 2

Leading edge tissue reaches anterior-most prospective neuroectoderm at equatorial region. Internal morphology of embryo at ACE. Blastopore is indicated by a black arrowhead. (A) Bombina orientalis at 4 h after blastopore appearance, (B) Rana japonica at 4 h after blastopore appearance, (C) Rana porosa brevipoda at 4 h after blastopore appearance, (D) Rana rugosa at 3 h after blastopore appearance, (E) Silurana tropicalis at 60 min after blastopore appearance, (F) Xenopus laevis at 3 h after blastopore appearance, (G) Bufo japonicus formosus at 4 h after blastopore appearance, (H) Bufo gargarizans miyakonis at 4 h after blastopore appearance, (I) Ambystoma mexicanum at 8 h after blastopore appearance, (J) Cynops  pyrrhogaster at 7 h after blastopore appearance, and (K) Hynobius nebulosus at 9 h after blastopore appearance in gastrula embryos. (A–F, I–K) The embryo was cut sagittally. The leading edge tissue is located in the equatorial region and the embryo has Brachet's cleft. (G, H) The embryo was fixed when Brachet's cleft was formed. The leading edge is located at the equatorial region. It may be important to mention that embryos of Urodele species have relatively long archenteron at anterior contact establishment (ACE), though anurans only have a dent at blastopore. This may be caused by a difference of gastrulation movement between them.

Figure 3

Vital dye staining of equatorial outer surface in Cynops pyrrhogaster. C. pyrrhogaster neurula with Nile blue staining of the equatorial surface at 7 h after blastopore appearance. The labeled region is indicated by a red arrowhead. (A) Immediately after labeling. (B) Labeled cells are localized in the anterior-most neural plate (15/25). Black arrowhead in panel A indicates blastopore.

Figure 4

Blastocoel roof after anterior contact establishment (ACE)is dispensable for neural formation. Tailbud embryos whose blastocoel roof was removed at the gastrula stage. Lateral view. Head is left. (A) Bombina orientalis, (B) Rana rugosa, (C) Xenopus laevis, (D) Ambystoma mexicanum, (E) Cynops pyrrhogaster, (F) Bufo japonicus formosus, and (G) Bufo gargarizans miyakonis. (A–E) The blastocoel roof was removed at ACE. Black arrows in A–C indicate cement gland. Broken line in (E) indicates the border of epidermis. (F, G) The blastocoel roof was removed 4 h after blastopore appearance when Brachet's cleft was just formed.

Figure 5

Formation of neural tissue is influenced by blastocoel roof removal time in Xenopus laevis. The blastocoel roof was removed 1, 2, and 3 h after blastopore appearance. The resultant embryos showed some differences during development to the tailbud stage. (A) The blastocoel roof was removed 1 h after blastopore appearance. The embryo has only a posterior dorsal axis. (B) The blastocoel roof was removed 2 h after blastopore appearance. The embryo has an anterior axis but no head. (C) The blastocoel roof was removed 3 h after blastopore appearance (ACE). The embryo has whole dorsal axis including cement gland.

Figure 6

Prospective neuroectoderm and lining mesoderm are continuously associated with each other during gastrulation in Cynops pyrrhogaster. (A) Schematic illustration of the experimental procedure for internal and external labeling. At the beginning, the external surface of the equatorial region was labeled with Nile blue 30 min before anterior contact establishment (ACE) (6.5 h after blastopore appearance), and then a bead was placed in the internal tissue at the same location as the labeled external surface, which can be seen from the internal side. (A) If the internal and the external tissues do not slide in tandem, the bead is under the labeled tissue at the end of gastrulation. (B) A labeled embryo 24 h after blastopore appearance (sagittal section). White bracket indicates the Nile blue labeled region. The bead in the internal tissue was found below the surface layer labeled with Nile blue. White arrowhead in panel B indicates blastopore.

Figure 7

Not dorsal lip but dorsal blastocoel floor acts as anterior organizer in normal development. (A) Schematic representation of blastocoel floor tracing experiments. (B, C) A bead was placed the inside corner of blastocoel at blastopore appearance, and the embryo was dissected sagittally at ACE in Xenopus laevis and Cynops pyrrhogaster, respectively. (D, E) A bead was placed with a small distance from the corner in X. laevis and C. pyrrhogaster, respectively. Arrowheads indicate the tip of Brachet's cleft. (F) Schematic representation of the grafting assay. The embryo at blastopore appearance is grafted ventrally with blastocoel floor tissue of anterior contact establishment (ACE) embryo, and allowed to develop to the tailbud stage. (G, H) X. laevis and C. pyrrhogaster tailbud embryos, respectively. The embryos form a secondary axis containing head structures, as indicated by white brackets. (I, J) The embryo is grafted ventrally with the dorsal lip (Spemann organizer) tissue of ACE embryo, and allowed to develop to the tailbud stage.

Figure 8

Expression patterns of chordin during early gastrulation in Xenopus laevis and Cynops pyrrhogaster. (A–E) Expression patterns of X. laevis chordin from late blastula to anterior contact establishment (ACE) (3 h after blastopore appearance). (F–J) Expression patterns of C. pyrrhogaster chordin from late blastula to ACE (7 h after blastopore appearance). (A, F) Expression in late blastula embryos. Chordin expression is first detected in the dorsal blastocoel roof. (B, G) Expression at the onset of gastrulation. Chordin is expressed not only in the surface layer (dorsal lip) but also internally in the blastocoel floor. (C, H) Expression in X. laevis 1 h after blastopore appearance, and in C. pyrrhogaster 2 h after blastopore appearance. A trench (red arrowheads) is formed between chordin-positive blastocoel floor and chordin-negative blastocoel roof. (D, I) Expression in X. laevis 2 h after blastopore appearance, and in C. pyrrhogaster 4 h after blastopore appearance. (E, J) Expression in ACE embryos (in X. laevis 3 h after blastopore appearance, and in C. pyrrhogaster 7 h after blastopore appearance). Insets are schematic drawings of the expression pattern. The developmental stages are shown at the bottom of each panel. Black arrowheads indicate blastopore. The outline of the embryo is shown by broken lines in panel (A). Note that the boundary between chordin-positive and -negative tissues corresponds to the bottom of the trench, and contributes to the tip of Brachet's cleft (red arrows).