Developmental analysis of fs(1)gastrulation defective,a dorsal-group gene of Drosophila melanogaster

Summary The gastrulation defective (gd) locus is a maternally expressed gene in Drosophila required for normal differentiation of structures along the embryonic dorso-ventral axis. Cuticular defects of the offspring from females with different combinations of gd alleles comprised a phenotypic continuum. Complementation among several alleles produced normal offspring while progressively more severe mutations produced a graded loss of structures from ventral, and then lateral, blastoderm cells. The most severely affected embryos consisted entirely of structures derived from dorsal blastoderm cells. Histological examination of staged siblings from selected allelic combinations showed that internal tissues were similarly affected. The tissues observed in amorphic embryos support new, more dorsal, assignments of fate map positions for blastoderm precursors of the cephalopharyngeal apparatus, hindgut and ventral nerve cord. The loss of ventral and lateral structures did not occur through cell death and appeared to involve a change in blastoderm cell fate. A direct effect of the mutations on blastoderm cell determination, however, was insufficient to explain the development of the dorsalized embryos. Intermediate phenotypes suggested that cell interactions or movements associated with morphogenesis are required for the determination of some cell fates in the dorsoventral axis. Thus, the developmental fate of all blastoderm cells may not be fixed at the time of blastoderm formation.     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage

Cell lineages during embryogenesis of the ascidian Halocynthia roretzi were analyzed up until the stage where each blastomere was fated to be only a single tissue type (i.e., the tissue restricted stage) by intracellular injection of horseradish peroxidase using the iontophoretic injection method. Initially, the developmental fates of all blastomeres of the 64-cell stage embryo were examined, and thereafter, only the fates of daughter blastomeres of those blastomeres that were not tissue restricted at the 64-cell stage were traced. The developmental fates of blastomeres were highly invariant except for two candidates for "equivalence groups" (J. Kimble, J. Sulston, and J. White (1979). In "Cell Lineage, Stem Cells and Cell Determination," pp. 59-68. Elsevier, Amsterdam/New York), in which cellular interaction is suggested to be involved in the specification of the fates. The right and left a8.25 cells gave rise to the otolith and ocellus, and the right and left b8.17 cells gave rise to the spinal cord and endodermal strand in a complementary manner. No fixed relationship existed between the position of the blastomere and its derivative. Most restrictions of cell fates occurred early in cleavage. The numbers of blastomeres which generated a single type of tissue were 44 at the 64-cell stage and 94 at the 110-cell stage. Eight pairs of blastomeres had not yet become tissue restricted by the 110-cell stage. Almost complete lineages of epidermis, nervous system, muscle, mesenchyme, notochord, and endodermal tissues were described, and a fate map was constructed for the blastula. For certain tissues, the primordial cells occupied two different regions. Supplementary investigations of the lineage of muscle cells were also performed on embryos of another species, Ciona intestinalis.     

Dorsal specification in blastoderm at the blastula stage in the goldfish, Carassius auratus

The teleost dorsoventral axis cannot be morphologically distinguished before gastrulation. Previous studies by the current authors have shown that localized dorsalizing activity in the yolk cell (YC) induces the dorsal tissues in the overlying blastoderm. In order to examine whether or not dorsal blastomeres are committed to their dorsal fate before the gastrula stage, a variety of transplant operations were performed in goldfish blastoderms at the mid- to late-blastula stages. When the blastoderm was cut from the YC, rotated horizontally at 180°, and recombined with the YC, the blastoderm frequently developed two axes, indicating that dorsal blastomeres of the blastula had already acquired the ability to differentiate into the organizer in the absence of dorsalizing signals from the YC. This result was further confirmed by experiments using ventralized embryos in which no dorsal structures formed: the axis formation was frequently observed in the normal blastoderm combined with the ventralized YC at the blastula stage. However, the axes formed in the absence of dorsal information from the YC exhibited a lower dorso-anterior index. Furthermore, the dorsal specification was not stably maintained when the dorsal cells were located far from the YC. These results suggest that the inductive and permissive influence of the YC may be required for the blastoderm to undergo full dorsal differentiation.     

One-eyed pinhead regulates cell motility independent of Squint/Cyclops signaling

In vertebrates, EGF-CFC factors are essential for Nodal signaling. Here, we show that the zygotic function of one-eyed pinhead, the zebrafish EGF-CFC factor, is necessary for cell movement throughout the blastoderm of the early embryo. During the blastula and gastrula stages, mutant cells are more cohesive and migrate slower than wild-type cells. Chimeric analysis reveals that these early motility defects are cell-autonomous; later, one-eyed pinhead mutant cells have a cell-autonomous tendency to acquire ectodermal rather than mesendodermal fates. Moreover, wild-type cells transplanted into the axial region of mutant hosts tend to form isolated aggregates of notochord tissue adjacent to the mutant notochord. Upon misexpressing the Nodal-like ligand Activin in whole embryos, which rescues aspects of the mutant phenotype, cell behavior retains the one-eyed pinhead motility phenotype. However, in squint;cyclops double mutants, which lack Nodal function and possess a more severe phenotype than zygotic one-eyed pinhead mutants, cells of the dorsal margin exhibit a marked tendency to widely disperse rather than cohere together. Elsewhere in the double mutants, for cells of the blastoderm and for rare cells of the gastrula that involute into the hypoblast, motility appears wild-type. Notably, cells at the animal pole, which are not under direct regulation by the Nodal pathway, behave normal in squint;cyclops mutants but exhibit defective motility in one-eyed pinhead mutants. We conclude that, in addition to a role in Nodal signaling, One-eyed pinhead is required for aspects of cell movement, possibly by regulating cell adhesion.     

Fate map for the 32-cell stage of Xenopus laevis

A complete fate map has been produced for the 32-cell stage of Xenopus laevis. Embryos with a regular cleavage pattern were selected and individual blastomeres were injected with the lineage label fluorescein-dextran-amine (FDA). The spatial location of the clones was deduced from three-dimensional (3D) reconstructions of later stages and the volume of each tissue colonized by labelled cells in each tissue was measured. The results from 107 cases were pooled to give a fate map which shows the fate of each blastomere in terms of tissue types, the composition of each tissue by blastomere, the location of each prospective region on the embryo and the fate of each blastomere in terms of spatial localization. Morphogenetic movements up to stage 10 (early gastrula) were assessed by carrying out a number of orthotopic grafts at blastula and gastrula stages using donor embryos uniformly labelled with FDA. Although there is a regular topographic projection from the 32-cell stage this varies a little between individuals because of variability of positions of cleavage planes and because of short-range cell mixing during gastrulation. The cell mixing means that the topographic projection fails for anteroposterior segments of the dorsal axial structures and it is not possible to include short segments of notochord or neural tube or individual somites on the pregastrulation fate map.     

Mitotic domains reveal early commitment of cells in Drosophila embryos

In embryos of Drosophila melanogaster all the nuclei in the syncytial egg divide with global synchrony during the first 13 mitotic cycles. But with cellularization in the 14th cycle, global mitotic synchrony ceases. Starting about one hour into the 14th interphase, at least 25 'mitotic domains', which are clusters of cells united by locally synchronous mitosis, partition the embryo blastoderm surface into a complex fine-scale pattern. These mitotic domains, which are constant from one embryo to the next, fire in the same temporal sequence in every embryo. Some domains consist of a single cell cluster straddling the ventral or dorsal midline. Most consist of two separate cell clusters that occupy mirror-image positions on the bilaterally symmetric embryo. Others comprise a series of members present not only as bilateral pairs but also as metameric repeats. Thus a domain can consist of either one, two, or many (if metamerically reiterated) clusters of contiguous cells. Within each cluster, mitosis starts in a single cell or in a small number of interior cells then spreads wave-like, in all directions, until it stops at the domain boundary. Each domain occupies a specific position along the anteroposterior axis--as determined by the expression pattern of the engrailed protein, and along the dorsoventral axis--as determined by cell count from the ventral midline. The primordia of certain larval structures appear to consist solely of the cells of one specific mitotic domain. Moreover, cells in at least some mitotic domains share specific morphogenetic traits, distinct from those of cells in adjacent domains. These traits include cell shape, spindle orientation, and participation by all the cells of a domain in an invagination. The specialized behaviors of the various mitotic domains transform the monolayer cell sheet of the blastoderm into the multilayered gastrula. I conclude that the fine-scale partitioning of the newly cellularized embryo into mitotic domains is an early manifestation of the commitment of cells to specific developmental fates.     

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.     

A revised fate map for amphioxus and the evolution of axial patterning in chordates

The chordates include vertebrates plus two groups of invertebrates(the cephalochordates and tunicates). Previous embryonic fatemaps of the cephalochordate amphioxus (Branchiostoma) were influencedby preconceptions that early development in amphioxus and ascidiantunicates should be fundamentally the same and that the earlyamphioxus embryo, like that of amphibians, should have ventralmesoderm. Although detailed cell lineage tracing in amphioxushas not been done because of limited availability of the embryosand because cleavage is radial and holoblastic with the blastomeresnearly equal in size and not tightly adherent until the mid-blastulastage, a compilation of data from gene expression and function,blastomere isolation and dye labeling allows a more realisticfate map to be drawn. The revised fate map is substantiallydifferent from that of ascidians. It shows (1) that the anteriorpole of the amphioxus embryo is offset dorsally from the animalpole only by about 20°, (2) that the ectoderm/mesendodermboundary (the future rim of the blastopore) is at the equatorof the blastula, which approximately coincides with the 3rdcleavage plane, and (3) that there is no ventral mesoderm duringthe gastrula stage. Involution or ingression of cells over theblastopore lip is negligible, and the blastopore, which is posterior,closes centripetally as if by a purse string. During the gastrulastage, the animal pole shifts ventrally, coming to lie about20° ventral to the anterior tip of the late gastrula/earlyneurula. Comparisons of the embryos of amphioxus and vertebratesindicate that in spite of large differences in the mechanicsof cleavage and gastrulation, anterior/posterior and dorsal/ventralpatterning occur by homologous genetic mechanisms. Therefore,the small, nonyolky embryo of amphioxus is probably a reasonableapproximation of the basal chordate embryo before the evolutionof determinate cleavage in the tunicates and the evolution largeamounts of yolk in basal vertebrates.     

The involvement of cAMP signaling pathway in axis specification in Xenopus embryos

The cAMP signaling system has been postulated to be involved in embryogenesis of many animal species, however, little is known about its role in embryonic axis formation in vertebrates. In this study, the role of the cAMP signaling pathway in patterning the body plan of the Xenopus embryo was investigated by expressing and activating the exogenous human 5-hydroxytryptamine type 1a receptor (5-HT(1a)R) which inhibits adenylyl cyclase through inhibitory G-protein in embryos in a spatially- and temporally-controlled manner. In embryos, ventral, but not dorsal expression and stimulation of this receptor during blastula and gastrula stages induced secondary axes but were lacking anterior structures. At the molecular level, 5-HT(1a)R stimulation induced expression of the dorsal mesoderm marker genes, and downregulated expression of the ventral markers but had no effect on expression of the pan mesodermal marker gene in ventral marginal zone explants. In addition, ventral expression and stimulation of the receptor partially restored dorsal axis of UV-irradiated axis deficient embryo. Finally, the total mass of cAMP differs between dorsal and ventral regions of blastula and gastrula embryos and this is regulated in a temporally-specific manner. These results suggest that the cAMP signaling system may be involved in the transduction of ventral signals in patterning early embryos.     

A default mechanism of spindle orientation based on cell shape is sufficient to generate cell fate diversity in polarised Xenopus blastomeres

The process of oriented divisions of polarised cells is a recurrent mechanism of cell fate diversification in development. It is commonly assumed that a specialised mechanism of spindle alignment into the axis of polarity is a prerequisite for such systems to generate cell fate diversity. Oriented divisions also take place in the frog blastula, where orientation of the spindle into the apicobasal axis of polarised blastomeres generates inner and outer cells with different fates. Here, we show that, in this system, the spindle orients according to the shape of the cells, a mechanism often thought to be a default. We show that in the embryo, fatedifferentiative, perpendicular divisions correlate with a perpendicular long axis and a small apical surface, but the long axis rather then the size of the apical domain defines the division orientation. Mitotic spindles in rounded, yet polarised, isolated Xenopus blastula cells orient randomly, but align into an experimentally introduced long axis when cells are deformed early in the cell cycle. Unlike other systems of oriented divisions, the spindle aligns at prophase, rotation behaviour is rare and restricted to small angle adjustments. Disruption of astral microtubules leads to misalignment of the spindle. These results show that a mechanism of spindle orientation that depends on cell shape rather than cortical polarity can nevertheless generate cell fate diversity from a population of polarised cells.     

Cell lineage of zebrafish blastomeres. III. Clonal analyses of the blastula and gastrula stages

Aspects of the early lineages of blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. Because of the optical clarity of the embryo, lineages of selected cells can be followed directly by microscopy through many cell divisions. Also, it is shown here that the fluorescent molecules fluorescein-dextran and rhodamine-horseradish peroxidase can be used as cell lineage tracers, marking the clonal progeny of founding blastomeres. The labeled cells can be easily visualized in the live embryo, and utilizing a sensitive video camera to amplify fluorescence, the same clone may be examined repeatedly while the cells divide and migrate. Cells that descend from a single blastomere remain closely associated together through the end of the blastula stage. At the time when epiboly begins (early gastrula) cells in the labeled clone scatter and become dispersed among unlabeled cells. It has been observed that there is no invariant mapping of the embryo's midline (determined by the position of the embryonic shield in the gastrula) with respect to the early planes of cleavage. This finding shows that in the zebrafish the region of the embryo that a cell will occupy is not specified by the cell's early ancestory.     

Role of the zebrafish trilobite locus in gastrulation movements of convergence and extension

Convergence and extension are gastrulation movements that participate in the establishment of the vertebrate body plan. Using new methods for quantifying convergence and extension movements of cell groups, we demonstrate that in wild-type embryos, dorsal convergence of lateral cells is initially slow, but speeds up between the end of the gastrula period and early segmentation. Convergence and extension movements of lateral cells in trilobite mutants are normal during the gastrula period but reduced by early segmentation. Morphometric studies revealed that during epiboly wild-type gastrulae become ovoid, whereas trilobite embryos remain rounder. By segmentation, trilobite embryos exhibit shorter, broader embryonic axes. The timing of these morphological defects correlates well with impaired cell movements, suggesting reduced convergence and extension are the main defects underlying the trilobite phenotype. Our gene expression, genetic, and fate mapping analyses show the trilobite mutation affects movements without altering dorsoventral patterning or cell fates. We propose that trilobite function is required for cell properties that promote increased speed of converging cells and extension movements in the dorsal regions of the zebrafish gastrula.     

Indeterminate cell lineage of the zebrafish embryo

We have examined the clonal progeny descended from individual blastomeres injected with lineage-tracer dye in the zebrafish embryo. Blastomeres arising by the same cleavages in different embryos generated clones in which the types and positions of cells were highly variable. Several features of early development were correlated with this diversity in cell fate. There was no fixed relationship between the plane of the first cleavage and the eventual plane of bilateral symmetry of the embryo. By blastula stages the cleavages of identified blastomeres were variable in pattern. Moreover, cell fate was not easily related to the longitudinal and dorsoventral position of the clone in the gastrula. These results establish that single blastomeres can potentially generate a highly diverse array of cell types and that the cell lineage is indeterminate.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary Some aspects of neural and epidermal cell lineages during embryogenesis of Drosophila melanogaster were studied by transplanting horseradish-peroxidase-(HRP-) labelled ectodermal cells from young gastrula donors into host embryos of similar ages. Heterotopic transplantations permitted us to assess the degree of commitment already attained by the transplanted cells. The resulting cell clones showed normal characteristics of cytodifferentiation and cell number. The results indicate that epidermal progenitors perform a maximum of three mitoses during embryonic development, whereas neuroblasts may perform more than ten mitoses. Clone size distribution is in both cases scattered, suggesting either a rather irregular mitotic pattern or cell death. As indicated by heterotopic transplantations, the neurogenic ectoderm for the ventral nervous system exhibits different neurogenic abilities in its different regions, decreasing from medial to lateral; we discuss the hypothesis that some medially located cells of the young gastrulating embryo could be committed towards the neural fate before segregating from the ectoderm. On the other hand, the cells of the dorsal ectodermal regions at the same stage seem to be indifferent with respect to commitment, for they are able to give rise to central neural lineages following their transplantation in the neurogenic region.     

Spemann's organizer--it's origin and derivatives (cellular-tissue and molecular-genetic aspects)

In 1924 H. Spemann and H. Mangold discovered that a piece of the dorsal lip of a blastopore from Triturus cristatus, after transplantation to the ventral side of another embryo, was able to cause the neighbouring tissues to change their fate and participate in the formation of a new embryo. The dorsal lip was termed "the organizer". Since then, for as long as 75 years, attempts have been made to establish the intimate mechanisms of the organizer activity. However, no real advance was achieved in their understanding. Within the last 15 years, genetic and molecular techniques have been vastly improved, to help in tracing the fate of many cell lineages, and in compiling more exactly the fate maps for different parts of the embryo. Using these data, I have attempted to trace the fate of Spemann's organizer after the early gastrula stage. Analysis of data on inductive abilities of the organizer cells, on the use of markers, and on the observation of expression of specific genes allowed to conclude that Spemann's organizer in amphibia and its homologues in other vertebrates too are heterogeneous: they are composed of distinct cell populations able to induce primarity the development of either the head or trunk parts of the embryo. These population, determined to become the head of the trunk organizers still at the blastula stage, may be located either in the single continuous cell layer (as in amphibia and birds) or separated among different tissue germs (as in mammals). When the dorsal-ventral orientation of the embryo is established and the organizer is switched on the very early invaginating cells of the dorsal blastopore lip (in the case of amphibia) move in advance of the entire invaginating mesoderm and by the end of gastrulation occupy the place just in front of the notochord. It is supposed that the early dorsal lip and the prechordal mesoderm (PCM) are one and the same cell population, i.e. during gastrulation Spemann's organizer transfers from the lip of blastopore to the prechordal zone. The PCM seems to play an exclusive role in the formation of a head in vertebrate, because some mutations in genes expressed in the PCM result in the entire head deletion. It is supposed that spreading of differentiating signals from the PCM occurs along the main body axis in both caudal and rostral directions. After the main body plan formation the PCM is replaced by adenohypophysis. This conclusion is drawn not only from the same topology of both these structures, but also from the similarities of a set of specific genetical markers expressed in these, that makes it possible to suppose the existence of deep connections and succession between them. The adenohypophysis seems to arise directly from the PCM, or cells of the ectoderm influenced by the PCM may be subsequently transformed into humoral cells of adenohypophysis. In this interpretation, adenohypophysis and the much earlier established PCM may be considered as derivatives of Spemann's organizer. This inference is supported by the fact that all the three above structures first originate in vertebrates only.     

Fate map and morphogenesis of presumptive neural crest and dorsal neural tube

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.     

Elucidating the origins of the vascular system: a fate map of the vascular endothelial and red blood cell lineages in Xenopus laevis.

Required to supply nutrients and oxygen to the growing embryo, the vascular system is the first functional organ system to develop during vertebrate embryogenesis. Although there has been substantial progress in identifying the genetic cascade regulating vascular development, the initial stages of vasculogenesis, namely, the origin of vascular endothelial cells within the early embryo, remain unclear. To address this issue we constructed a fate map for specific vascular structures, including the aortic arches, endocardium, dorsal aorta, cardinal veins, and lateral abdominal veins, as well as for the red blood cells at the 16-cell stage and the 32-cell stage of Xenopus laevis. Using genetic markers to identify these cell types, our results suggest that vascular endothelial cells can arise from virtually every blastomere of the 16-cell-stage and the 32-cell-stage embryo, with different blastomeres preferentially, though not exclusively, giving rise to specific vascular structures. Similarly, but more surprisingly, every blastomere in the 16-cell-stage embryo and all but those in the most animal tier of the 32-cell-stage embryo serve as progenitors for red blood cells. Taken together, our results suggest that during normal development, both dorsal and ventral blastomeres contribute significantly to the vascular endothelial and red blood cell lineages.