Regional specification of the head and trunk-tail organizers of a urodele (Cynops pyrrhogaster) embryo is patterned during gastrulation

The dorsal marginal zone (DMZ) of an amphibian early gastrula is thought to consist of at least two distinct domains: the future head and trunk-tail organizers. We studied the mechanism by which the organizing activities of the lower half of the DMZ (LDMZ) of the urodelean (Cynops pyrrhogaster) embryo are changed. The uninvoluted LDMZ induces the notochord and then organizes the trunk-tail structures, whereas after cultivation in vitro or suramin treatment, the same LDMZ loses the notochord-inducing ability and organizes the head structures. A cell-lineage experiment indicated that the change in the organizing activity of the LDMZ was reflected in the transformation of the inductive ability: from notochord-inducing to neural-inducing activity. Using RT-PCR, we showed that the LDMZ expressed gsc, lim-1, chordin, and noggin, but not the mesoderm marker bra. In the sandwich assay, the LDMZ induced bra expression in the animal cap ectoderm, but the inductive activity was inhibited by cultivation or suramin treatment. The present study indicates that the change in the organizing activity of the LDMZ from trunk-tail to head is coupled with the loss of notochord-inducing activity. Based on these results, we suggest that this change is essential for the specification of the head and trunk-tail organizers during gastrulation.     

Blastopore formation and dorsal mesoderm induction are independent events in early Cynops embryogenesis

The independent roles of blastopore formation and dorsal mesoderm induction in dorsal axis formation of the Cynops pyrrhogaster embryo were attempted to be clarified. The blastopore-forming (bottle) cells originated mainly from the progeny of the mid-dorsal C and/or D blastomeres of the 32-cell embryo, but were not defined to a fixed blastomere. It was confirmed that the isolated dorsal C and D blastomeres autonomously formed a blastopore. Ultraviolet-irradiated eggs formed an abnormal blastopore and then did not form a dorsal axis, although the lower dorsal marginal zone (LDMZ) still had dorsal mesoderm-inducing activity. Involution of the dorsal marginal zone was disturbed by the abnormal blastopore. These embryos were rescued by artificially facilitating involution of the dorsal marginal zone. Suramin-injected and nocodazole-treated blastulae did not have involution of the dorsal marginal zone, although the blastopore was formed. Neither embryos formed the dorsal axis. The dorsal mesoderm-inducing activity of the LDMZ in the nocodazole-treated gastrulae was still active. In contrast, the LDMZ of the suramin-injected embryos lost its dorsal mesoderm-inducing activity. bra expression was activated in the nocodazole-treated embryos but not in the suramin-injected embryos. The present study suggested that (i) the dorsal determinants consist of blastopore-forming and dorsal mesoderm-inducing factors, which are not always mutually dependent; (ii) both factors are activated during the late blastula stage; (iii) the dorsal marginal zone cannot specify to an organized notochord and muscle without the involution that blastopore formation leads to; and (iv) the localization of both factors in the same place is prerequisite for dorsal axis formation.     

Origin of the prechordal plate and patterning of the anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster

We analyzed the notochord formation, formation of the prechordal plate, and patterning of anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster. The lower (LDMZ) and upper (UDMZ) domains of the dorsal marginal zone (DMZ) of the early gastrula involuted and formed two distinct domains: the anterior fore-notochordal endodermal roof and the posterior domain containing the prospective notochord. Cygsc is expressed in the LDMZ from the onset of gastrulation, and the Cygsc-expressing LDMZ planarly induces the notochord in the UDMZ at the early to mid gastrula stages. At the mid to late gastrula stages, part of the Cygsc-expressing LDMZ is confined to the prechordal plate. On the other hand, Cybra expression only begins at mid gastrula stage, coincident with notochord induction at this stage. Anteroposterior regional specificity of the neural plate was patterned by the posterior domain of the involuting archenteron roof containing the prospective notochord at the mid to late gastrula stages. Cynops gastrulation thus differs significantly from Xenopus gastrulation in that the regions of the DMZ are specified from the onset of gastrulation, while the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Thus we propose that Cynops gastrulation is divided into two phases: a notochord induction phase in the early to mid gastrula, and a neural induction phase in the mid to late gastrula.     

Formation of the dorsal marginal zone in Xenopus laevis analyzed by time-lapse microscopic magnetic resonance imaging

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.     

F-Actin is a marker of dorsal induction in earlyPatella embryos

Summary The dorsal-ventral axis inPatella vulgata embryos is established at the 32-cell stage by an inductive interaction between the animal micromeres and one vegetal macromere. This vegetal macromere, once induced, is called the 3D macromere, and marks the future dorsal side of the embryo. We examined the pattern of filamentous (F) actin in such embryos using fluorescent phalloidin and found that this dorsal 3D macromere contains more F-actin than the remainder of the cells. In addition, only one of its two daughter cells, i.e. the 4D macromere, retains this higher density. In embryos in which the establishment of the dorsal-ventral axis has been experimentally inhibited via treatment with monensin, such differences in F-actin were not found. These results suggest that the appearance of an increased density of F-actin in the dorsal 3D and 4D macromeres of normal embryos requires the inductive interactions that establish the dorsal-ventral axis. We therefore conclude that F-actin is an early marker for dorsal induction in thePatella embryo.     

东方蝾螈原肠形成期瓶状细胞的结构及其对胚胎发育的影响

以１００只东方蝾螈早期胚胎为材料,用ＳＥＭ和ＴＥＭ对瓶状细胞形态结构和除去瓶状细胞的胚胎进行观察,去除瓶状细胞后,胚胎的原肠形成不会中断,外包和内卷仍能正常进行,并可以发育成幼螈,但有部分胚胎原肠前端缺失,长期培养的蝾螈胚对针刺激有反应,幼虫阶段中自由活动,我们的结论是：１）东方蝾螈原肠胚的瓶状细胞除了原肠前端存在外,还有部分瓶状细胞与原肠长轴垂直,从力学角度推断瓶状细胞在原肠形成过程中不起主要作     

Direct evidence of an essential role for extended involution in the specification of a dorsal marginal mesoderm during Cynops gastrulation

It has been indicated that specification of the dorsal marginal mesoderm of the Cynops gastrula is established by vertical interactions with other layers, which occur during its extended involution. In the present study, when the prospective notochordal area of the early gastrula was almost completely removed together with the dorsal mesoderm-inducing endoderm and most of the bottle cells, the D-less gastrulas still formed the dorsal axis with a well-differentiated notochord; in half of them, where the involution occurred bi-laterally, twin axes were observed. On the other hand, when the wound of a D-less gastrula was repaired by transplanting the ventral marginal zone and ectoderm, the formation of the dorsal axis was inhibited if the involution of the lateral marginal zone was prevented by the transplanted piece. The present study suggests that: (i) cells having dorsal mesoderm-forming potency distribute farther laterally than the fate map; and (ii) the extended involution plays an essential role in the specification of the dorsal marginal mesoderm, especially in notochordal differentiation in normal Cynops embryogenesis.     

Experimental analysis of the extension of the dorsal marginal zone in Pleurodeles waltl gastrulae

The capacity for extension of the dorsal marginal zone (DMZ) in Pleurodeles waltl gastrulae was studied by scanning electron microscopy and grafting experiments. At the onset of gastrulation, the cells of the animal pole (AP) undergo important changes in shape and form a single layer. As gastrulation proceeds, the arrangement of cells also changes in the noninvoluted DMZ: radial intercalation leads to a single layer of cells. Grafting experiments involving either AP or DMZ explants were performed using a cell lineage tracer. When rotated 90 degrees or 180 degrees, grafted DMZ explants were able to involute normally and there was extension according to the animal-vegetal axis of the host. In contrast, neither single nor bilayered explants from AP involutes completely, and neither extends when grafted in place of the DMZ. Furthermore, when inside of the host, these AP grafts curl up and inhibit the closure of the blastopore. Once transplanted to the AP region, the DMZ showed no obvious autonomous extension. DMZs cultured in vitro showed little extension and this only from the late gastrula stage onward. Removal of blastocoel roof blocked involution to a varied extent, depending on the developmental stage of the embryos. From these results, it is argued that differences could well exist in the mechanism of gastrulation between anuran and urodele embryos. That migrating mesodermal cells play a major role in urodele gastrulation is discussed.     

A cytoplasmic determinant for dorsal axis formation in an early embryo of Xenopus laevis

In Xenopus laevis, dorsal cells that arise at the future dorsal side of an early cleaving embryo have already acquired the ability to cause axis formation. Since the distribution of cytoplasmic components is markedly heterogeneous in an egg and embryo, it has been supposed that the dorsal cells are endowed with the activity to form axial structures by inheriting a unique cytoplasmic component or components localized in the dorsal region of an egg or embryo. However, there has been no direct evidence for this. To examine the activity of the cytoplasm of dorsal cells, we injected cytoplasm (dorsal cytoplasm) from dorsal vegetal cells of a Xenopus 16-cell embryo into ventral vegetal cells of a simultaneous recipient. The cytoplasm caused secondary axis formation in 42% of recipients. Histological examination revealed that well-developed secondary axes included notochord, as well as a neural tube and somites. However, injection of cytoplasm of ventral vegetal cells never caused secondary axis and most recipients became normal tailbud embryos. Furthermore, about two-thirds of ventral isolated halves injected with dorsal cytoplasm formed axial structures. These results show that dorsal, but not ventral, cytoplasm contains the component or components responsible for axis formation. This can be the first step towards identifying the molecular basis of dorsal axis formation.     

Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is homologous to the vertebrate proto-oncogene c-rel. In wild-type embryos, the dorsal protein is found in the cytoplasm during cleavage. After the nuclei migrate to the periphery of the embryo, a ventral-to-dorsal gradient of nuclear dorsal protein is established. The formation of the nuclear gradient is disrupted in mutant embryos from other maternally active dorsal-ventral polarity genes: in dorsalized embryos only cytoplasmic protein is observed, while in ventralized embryos the nuclear gradient is shifted dorsally. My findings suggest that nuclear localization is critical for dorsal to function as a morphogen and that the distribution of the dorsal protein determines cell fate along the dorsal-ventral axis.     

Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid

Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.     

Role of BMP-4 in the inducing ability of the head organizer in Xenopus laevis

BMP-4 has been implicated in the patterning of the Dorsal-Ventral axis of mesoderm and ectoderm. In this study, we describe the posteriorizing effect of BMP-4 on the neural inducing ability of dorsal mesoderm (dorsal lip region) in Xenopus gastrulae. Dorsal lip explants dissected from stage 10.25 embryos retained anterior inducing ability when precultured for 6 hrs until sibling embryos reach stage 12. When the dorsal lips from stage 10.25 embryos were treated with a range of BMP-4 concentrations, posterior tissues were induced in adjacent ectoderm in a dose-dependent manner. Thus activin-treated explants able to act as head inducers can also induce posterior structures in the presence of BMP-4. To investigate whether BMP-4 directly affects the inducing ability of dorsal mesoderm, we blocked the BMP-4 signaling pathway by injection of mRNA encoding a truncated form of the BMP-4 receptor (tBR) mRNA. Under these conditions, activin-treated explants induced anterior tissues following BMP-4 treatment. Taken together, these results indicate that BMP-4 may affect the head inducing ability of dorsal mesoderm and confer trunk-tail inducing ability during Xenopus gastrulation.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

Gastrulation and pre-gastrulation morphogenesis, inductions, and gene expression: similarities and dissimilarities between urodelean and anuran embryos

Studies of meso-endoderm and neural induction and subsequent body plan formation have been analyzed using mainly amphibians as the experimental model. Xenopus is currently the predominant model, because it best enables molecular analysis of these induction processes. However, much of the embryological information on these inductions (e.g., those of the Spemann-Mangold organizer), and on the morphogenetic movements of inductively interacting tissues, derives from research on non-model amphibians, especially urodeles. Although the final body pattern is strongly conserved in vertebrates, and although many of the same developmental genes are expressed, it has become evident that there are individually diverse modes of morphogenesis and timing of developmental events. Whether or not this diversity represents essential differences in the early induction processes remains unclear. The aim of this review is to compare the gastrulation process, induction processes, and gene expressions between a urodele, mainly Cynops pyrrhogaster, and an anura, Xenopus laevis, thereby to clarify conserved and diversified aspects. Cynops gastrulation differs significantly from that of Xenopus in that specification of the regions of the Xenopus dorsal marginal zone (DMZ) are specified before the onset of gastrulation, as marked by blastopore formation, whereas the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Detailed comparison of the germ layer structure and morphogenetic movements during the pre-gastrula and gastrula stages shows that the entire gastrulation process should be divided into two phases of notochord induction and neural induction. Cynops undergoes these processes sequentially after the onset of gastrulation, whereas Xenopus undergoes notochord induction during a series of pre-gastrulation movements, and its traditionally defined period of gastrulation only includes the neural induction phase. Comparing the structure, fate, function and state of commitment of each domain of the DMZ of Xenopus and Cynops has revealed that the true form of the Spemann-Mangold organizer is suprablastoporal gsc-expressing endoderm that has notochord-inducing activity. Gsc-expressing deep endoderm and/or superficial endoderm in Xenopus is involved in inducing notochord during pre-gastrulation morphogenesis, rather than both gsc- and bra-expressing tissues being induced at the same time.     

Experimental analysis of gravitational effects on amphibian gastrulation.

The effects of simulated microgravity on blastopore (Bp) formation were analysed in Xenopus laevis and Cynops pyrrhogaster embryos. Simulated microgravity produced by clinostat rotation shifted the Bp-forming region toward the vegetal pole, more markedly in Cynops embryos than in Xenopus embryos. The simulated microgravity induced aggregation of endoderm cells at the center of the embryo and separation between the endoderm and presumptive mesoderm (PM). These findings suggest that clinostat treatment disrupts cell-to-cell interaction between endoderm and PM by increasing the separation between them and, as a result, Bp formation may be shifted towards the vegetal pole.     

Evolution of the organizer and the chordate body plan

The discovery of the organizer by Spemann and Mangold in 1924 raised two kinds of questions: those about the means of patterning the chordate body axis and those about the mechanisms of cell determination by induction. Some researchers, stressing the second, have suggested over the years that the organizer is poorly named and doesn't really organize because inducers act permissively, because they are not unique to the organizer, and because multipotent responsive cells develop complex local differentiations under artificial conditions. Furthermore, with the discovery of meso-endoderm induction in 1969, the possibility arose that this earlier induction generates as much organization as, or more than, does the organizer itself. Evidence is summarized in this article that the organizer does fulfill its title with regard to pattern formation: it adds greatly to embryonic organization by providing information about time, place, scale, and orientation for development by nearby members of the large multipotent competence groups surrounding the organizer. Embryos having smaller or larger organizers due to experimental intervention develop defective axial organization. Without an organizer the embryo develops no body axis and none of the four chordate characters: the notochord, gill slits, dorsal hollow nerve chord, and post-anal tail. For normal axis formation, the organizer's tripartite organization is needed. Each part differs in inducers, morphogenesis, and self-differentiation. The organizer is a trait of development of all members of the chordate phylum. In comparison to hemichordates, which constitute a phylum with some similarities to chordates, the chordamesoderm part is unique to the chordate organizer (the trunk-tail organizer). Its convergent extension displaces the gastrula posterior pole from alignment with the animal-vegetal axis and generates a new anteroposterior axis orthogonal to this old one. Once it has extended to full length, its signaling modifies the dorsoventral dimension. This addition to the organizer is seen as a major event in chordate evolution, bringing body organization beyond that achieved by oocyte organization and meso-endoderm induction in other groups.     

A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is determined by a morphogen gradient established by the action of 12 maternal-effect genes: the dorsal group genes and cactus. One of the dorsal group genes, dorsal (dl), encodes the putative morphogen. Although no overall asymmetry in the distribution of dorsal protein is observed, a gradient of nuclear concentration of dl protein is established during cleavage stages, with a maximum at the ventral side of the egg. At the dorsal side of the egg, the protein remains in the cytoplasm. Nuclear localization of the dl protein, and hence gradient formation, is blocked in dorsalizing alleles of all of the other dorsal group genes, while in ventralizing mutants nuclear localization extends to the dorsal side of the egg. A correlation between dl protein distribution and embryonic pattern in mutant embryos indicates that the nuclear concentration of the dl protein determines pattern along the dorsoventral axis.     

Formation of germ layers and roles of the dorsal lip of the blastopore in normally developing embryos of the newt Cynops pyrrhogaster

Three-dimensional relationships between tissues during the formation of germ layers were studied in sections of normally developing embryos of the newt, Cynops pyrrhogaster. In gastrulae, the inner postinvolution layer was not in direct contact with the outer preinvolution layer as a result of the presence of an intervening layer of cells. Only after the formation of the yolk plug, a narrow strip of primitive notochord, which consisted of columnar cells, established a close contact with the central part of the overlaying presumptive neural plate. The primitive notochord was also linked to endoderm at its right and left margins, facing the archenteron. Mesodermal cells other than notochord cells were mesenchymal until the neurula stage, when primitive somites appeared on both sides of the notochord. From a comparison of the relative locations of tissues in embryos at different stages of development, it was shown that the notochord elongates by a remodeling of the mass of the primitive notochord, and that, as the anteriorly directed translocation of the neural area and the invagination of endoderm occur, these processes keep pace with the elongation of the notochord. These observations suggest organizing or guiding roles for the notochord in the formation of germ layers. A role for the dorsal lip of the blastopore as the organizer is discussed in relation to the origin of the notochord.