鸡胚原肠胚期原条细胞命运决定于其所处的局部微环境

在果蝇、斑马鱼、鸡等三胚层动物胚胎早期发育的原肠胚期,原条两侧的上胚层细胞进入原条经历上皮-间充质转化(EMT),迁移进入囊胚腔,形成松散的中胚层细胞,位于原条不同部位的细胞其迁移路线和分化命运不同,如前部原条细胞贡献于体节和心脏等,而后部原条细胞则迁移至胚外形成血岛。为了研究细胞的迁移途径及分化命运是否会随着细胞所处不同部位微环境的改变而改变,利用传统的移植技术,将宿主鸡胚原条前部的一部分细胞用GFP阳性的相同时期鸡胚原条组织替换,培养一段时间后,用荧光体视显微镜追踪GFP阳性细胞的迁移途径。结果发现,从供体原条后部移植到宿主原条前部的细胞遵循原条前部细胞迁移的路线,反之亦然;原位杂交结果显示移植后的GFP阳性细胞分化为所处部位的细胞类型。上述结果表明:鸡胚原肠胚期原条细胞迁移和分化的命运决定于细胞所处的微环境或者说局部基因表达的时空性。     

PTEN抑制胚胎原肠胚形成期EMT的过程

PTEN(Phosphatase and tensin homolog)是一种重要的抑癌基因,具有非常广泛的生物学活性,例如在细胞的生长发育、迁移、凋亡和信号传导等均发挥重要作用。PTEN基因表达始于在胚胎早期的上胚层,而后主要出现在神经外胚层和胚胎中胚层结构,表明PTEN可能参与胚胎早期发育过程的细胞迁移、增殖和分化。文章主要应用在体改变早期胚胎PTEN的表达水平来观察其对上胚层至中胚层细胞转换—EMT(Epithe-lial-mesenchymal transition)的作用。首先,原位杂交结果提示,内源性PTEN表达在原条以及之后的中胚层细胞结构如体节等。在体PTEN转染实验,体外培养至HH3期的鸡胚胎,转染Wt PTEN-GFP或移植Wt PTEN-GFP原条组织至未转染的同时期的宿主胚胎相同部位后,观察到PTEN转染细胞大都由上胚层迁移至原条并滞留于原条,不再参与中胚层细胞形成。移植实验也得到相似结果,发现在Wt PTEN-GFP阳性原条组织移植后很少细胞迁移出原条。另外在原肠胚期PTEN siRNA降调胚胎一侧PTEN基因后,降调侧中胚层细胞数明显少于正常侧。上述研究结果均提示PTEN基因在胚胎原肠胚期三胚层形成过程中可能具有抑制EMT的作用。     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

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

Gastrulation in the amniote begins with the formation of a primitive streak through which precursors of definitive mesoderm and endoderm ingress and migrate to their embryonic destinations. This organizing center for amniote gastrulation is induced by signal(s) from the posterior margin of the blastodisc. The mode of action of these inductive signal(s) remains unresolved, since various origins and developmental pathways of the primitive streak have been proposed. In the present study, the fate of chicken blastodermal cells was traced for the first time in ovo from prestreak stages XI-XII through HH stage 3, when the primitive streak is initially established and prior to the migration of mesoderm. Using replication-defective retrovirus-mediated gene transfer and vital dye labeling, precursor cells of the stage 3 primitive streak were mapped 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 seen from the anterolateral epiblast. Instead, the precursor cells generated daughter cells that underwent a polarized cell division oriented perpendicular to the anteroposterior embryonic axis. The resulting daughter cell population was arranged in a longitudinal array extending the complete length of the primitive streak. Furthermore, expression of cVg1, a posterior margin-derived signal, at the anterior marginal zone induced adjacent epiblast cells, but not those lateral to or distant from the signal, to form an ectopic primitive streak. The cVg1-induced epiblast cells also exhibited polarized cell divisions during ectopic primitive streak formation. These results suggest that blastoderm cells located immediately anterior to the posterior marginal zone, which secretes an inductive signal, undergo spatially directed cytokineses during early primitive streak formation.     

Control of early anterior-posterior patterning in the mouse embryo by TGF-beta signalling

Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissue layers. Positioning of the future anterior-posterior axis of the embryo occurs through coordinated cell movements that rotate a pre-existing proximal-distal (P-D) axis. Overt axis formation becomes evident when a discrete population of proximal epiblast cells become induced to form mesoderm, initiating primitive streak formation and marking the posterior side of the embryo. Over the next 12-24 h the primitive streak gradually elongates along the posterior side of the epiblast to reach the distal tip. The most anterior streak cells comprise the 'organizer' region and include the precursors of the so-called 'axial mesendoderm', namely the anterior definitive endoderm and prechordal plate mesoderm, as well as those cells that give rise to the morphologically patent node. Signalling pathways controlled by the transforming growth factor-beta ligand nodal are involved in orchestrating the process of axis formation. Embryos lacking nodal activity arrest development before gastrulation, reflecting an essential role for nodal in establishing P-D polarity by generating and maintaining the molecular pattern within the epiblast, extraembryonic ectoderm and the visceral endoderm. Using a genetic strategy to manipulate temporal and spatial domains of nodal expression reveals that the nodal pathway is also instrumental in controlling both the morphogenetic movements required for orientation of the final axis and for specification of the axial mesendoderm progenitors.     

The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.     

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

Knowledge of the molecular mechanisms regulating cell ingression, epithelial–mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.     

The type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral head development in mice.

The type II activin receptors, ActRIIA and ActRIIB, have been shown to play critical roles in axial patterning and organ development in mice. To investigate whether their function is required for mesoderm formation and gastrulation as implicated in Xenopus studies, we generated mice carrying both receptor mutations by interbreeding the ActRIIA and ActRIIB knockout mutants. We found that embryos homozygous for both receptor mutations were growth arrested at the egg cylinder stage and did not form mesoderm. Further analyses revealed that ActRIIA(-/-)ActRIIB(+/-) and about 15% of the ActRIIA(-/-) embryos failed to form an elongated primitive streak, resulting in severe disruption of mesoderm formation in the embryo proper. Interestingly, we observed similar gastrulation defects in ActRIIA(-/-)nodal(+/-) double mutants, which, if they developed beyond the gastrulation stage, displayed rostral head defects and cyclopia. These results provide genetic evidence that type II activin receptors are required for egg cylinder growth, primitive streak formation, and rostral head development in mice.     

FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

Axis specification and morphogenesis in the mouse embryo require Nap1, a regulator of WAVE-mediated actin branching

Dynamic cell movements and rearrangements are essential for the generation of the mammalian body plan, although relatively little is known about the genes that coordinate cell movement and cell fate. WAVE complexes are regulators of the actin cytoskeleton that couple extracellular signals to polarized cell movement. Here, we show that mouse embryos that lack Nap1, a regulatory component of the WAVE complex, arrest at midgestation and have defects in morphogenesis of all three embryonic germ layers. WAVE protein is not detectable in Nap1 mutants, and other components of the WAVE complex fail to localize to the surface of Nap1 mutant cells; thus loss of Nap1 appears to inactivate the WAVE complex in vivo. Nap1 mutants show specific morphogenetic defects: they fail to close the neural tube, fail to form a single heart tube (cardia bifida), and show delayed migration of endoderm and mesoderm. Other morphogenetic processes appear to proceed normally in the absence of Nap1/WAVE activity: the notochord, the layers of the heart, and the epithelial-to-mesenchymal transition (EMT) at gastrulation appear normal. A striking phenotype seen in approximately one quarter of Nap1 mutants is the duplication of the anteroposterior body axis. The axis duplications arise because Nap1 is required for the normal polarization and migration of cells of the Anterior Visceral Endoderm (AVE), an early extraembryonic organizer tissue. Thus, the Nap1 mutant phenotypes define the crucial roles of Nap1/WAVE-mediated actin regulation in tissue organization and establishment of the body plan of the mammalian embryo.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Pten regulates collective cell migration during specification of the anterior-posterior axis of the mouse embryo

Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.     

Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.

The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.     

The organizer of the mouse gastrula is composed of a dynamic population of progenitor cells for the axial mesoderm

An organizer population has been identified in the anterior end of the primitive streak of the mid-streak stage embryo, by the expression of Hnf3beta, Gsc(lacZ) and Chrd, and the ability of these cells to induce a second neural axis in the host embryo. This cell population can therefore be regarded as the mid-gastrula organizer and, together with the early-gastrula organizer and the node, constitute the organizer of the mouse embryo at successive stages of development. The profile of genetic activity and the tissue contribution by cells in the organizer change during gastrulation, suggesting that the organizer may be populated by a succession of cell populations with different fates. Fine mapping of the epiblast in the posterior region of the early-streak stage embryo reveals that although the early-gastrula organizer contains cells that give rise to the axial mesoderm, the bulk of the progenitors of the head process and the notochord are localized outside the early gastrula organizer. In the mid-gastrula organizer, early gastrula organizer derived cells that are fated for the prechordal mesoderm are joined by the progenitors of the head process that are recruited from the epiblast previously anterior to the early gastrula organizer. Cells that are fated for the head process move anteriorly from the mid-gastrula organizer in a tight column along the midline of the embryo. Other mid-gastrula organizer cells join the expanding mesodermal layer and colonize the cranial and heart mesoderm. Progenitors of the trunk notochord that are localized in the anterior primitive streak of the mid-streak stage embryo are later incorporated into the node. The gastrula organizer is therefore composed of a constantly changing population of cells that are allocated to different parts of the axial mesoderm.     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

Shaping and bending of the avian neural plate as analysed with a fluorescent-histochemical marker

Shaping and bending of the neural plate are cardinal events of neurulation. These processes are initiated in avian embryos shortly after the onset of gastrulation and concluded concomitantly with the completion of gastrulation. The epiblast undergoes extensive morphogenetic movements during gastrulation and neurulation, but the directions, distances, rates, mechanisms and roles of such rearrangements are largely unknown. To begin to understand these morphogenetic movements, we have mapped regional displacements of the epiblast by injecting a fluorescent-histochemical marker into selected prenodal, nodal and postnodal levels of the blastoderm. Lateral epiblast regions (600 microns lateral to the midline and consisting primarily of surface epithelium) are displaced craniomedially, medial regions (300 microns lateral to the midline and consisting of neural plate and preingressed mesoderm) predominantly medially, and midline regions (consisting of neural plate and primitive streak) predominantly caudally. Displacements within the avian neural plate parallel those previously described for the amphibian neural plate. Furthermore, similar tissue displacements occur within the prenodal and postnodal levels of the avian epiblast despite the fact that neurulation is occurring in the former and gastrulation in the latter. Finally, our results show that ectodermal rudiments contained within a single cross-sectional level of the embryo are a composite of cells derived from multiple craniocaudal and mediolateral levels. Thus, regional tissue displacements are important events to consider in the analysis of the early morphogenesis of axial and paraxial organ rudiments derived from the epiblast.     

Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes

Previous studies have shown that medaka primordial germ cells (PGC) are first distinguishable by olvas expression during late gastrulation, and that they migrate to the gonadal region through the lateral plate mesoderm. Here, we demonstrate that medaka nanos expression marks the germ line at early gastrulation stage. By marking the germ line with green fluorescent protein (GFP) fused to the nanos 3' untranslated region, we were able to visualize the behavior of PGC using time-lapse imaging. We show that there are three distinct modes of PGC migration that function at different stages of development. At early gastrulation stage, PGC actively migrate towards the marginal zone, a process that requires the function of a chemokine receptor, CXCR4. However, at late gastrulation stage, PGC change the mode and direction of their movement, as they are carried towards the midline along with somatic cells undergoing convergent movements. After aligning bilaterally, PGC actively migrate to the posterior end of the lateral plate mesoderm. This posterior movement depends on the activity of both HMGCoAR and a ligand of CXCR4, SDF-1a. These results demonstrate that PGC undergo different modes of migration to reach the prospective gonadal region of the embryo.     

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.     

Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes

SUMMARY. The embryonic development of germ cells in tetrapods is described, focusing on groups with the inductive mode of germ cell specification. In mammals PGCs are induced early in the gastrulation process, they are internalized with future extraembryonic mesoderm in the early posterior primitive streak, and specified soon thereafter. Strong evidence indicates that a similar process occurs in turtles and some other reptiles. In amniotes, the PGCs appear well before formation of the gonad in the posterior trunk, resulting in a period in which they are located outside the embryo before their migration to the gonad. In contrast, in urodeles the PGCs appear relatively late, and throughout development maintain a position close to precursors of the somatic cells of the gonad so that migration is not required. In lampreys early development of germ cells is strikingly similar to that in urodeles, suggesting this is the primitive process. As amniotes evolved large yolky eggs and better access to nutrition, development of the posterior half of the trunk became more dependent on cell proliferation; this was followed or accompanied by a shift of early germ cell development to the equivalent of the early primitive streak. A similar process may have occurred as some basal vertebrates developed large yolky eggs.