Establishment of the scleral cartilage in the chick.

This study was undertaken to investigate the establishment of the scleral cartilage in the chick embryo. Johnston et al. (1974) has demonstrated that most of the cells of the scleral cartilage originate in the cranial neural crest. By means of a series of chorioallantoic grafts of pigmented retina, and its adherent periocular mesenchyme from stage 11 to 25, the present experiments show that the cranial neural crest cells arrive at the eye in sufficient numbers to form cartilage by stage 14. Pigmented retina, denuded of mesenchyme, from stage 16 embryos implanted into the head of stage 13 embryos induces cartilage formation in head mesenchyme. However, neither pigmented retina nor spinal cord could induce cartilage formation in chorioallantoic mesenchyme. Combination grafts of cranial neural crest and presumptive optic vesicle developed neural tissue, pigmented retina, and in some cases sclera-like cartilage. Thus, periorbital mesenchyme, derived largely from cranial neural crest, at about stage 14 develops the scleral cartilage in response to induction by the pigmented retina.     

Expression of Otx homeodomain proteins induces cell aggregation in developing zebrafish embryos

In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. We demonstrate here that Otx proteins have a dramatic effect on cell-cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila otd RNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, we demonstrate that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain.     

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.     

Investigations into the degree of cell mixing that occurs between the 8-cell stage and the blastocyst stage of mouse development.

This study was designed to assess the degree of cell mixing that occurs during the early development of the mouse embryo, and thus provide information which is important in relation to the current theories of differentiation. Previous studies of this nature have involved either chimeric composites, or have only followed a very limited number of cells in the embryo. Here the products of one of the 4-cell stage blastomeres have been labeled with tritiated thymidine, at a level which allows their descendants to be identified three or four cell divisions later, and recombined with the remaining blastomeres of the same embryo. After fixing and sectioning of the embryos at the blastocyst stage the locations of the labelled cells have been analyzed to assess the degree of clumping that they display. A significant tendency for the products of this one 4-cell stage blastomere to be confined to a single area in the blastocyst is demonstrated. This indicates that there is little marked cell movement during the observation period. The relevance of these results to current knowledge of blastocyst development is discussed.     

Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector

The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos. © 1995 Wiley-Liss, Inc.     

Slow intermixing of cells during Xenopus embryogenesis contributes to the consistency of the blastomere fate map

The relatively consistent fates of the blastomeres of the frog embryo could result from (i) predetermination of the blastomeres or (ii) reproducible morphogenetic cell movements. In some species, the mixing of the cells during development provides a test between these alternative hypotheses. If blastomeres are predetermined, then random intermixing of the descendants with neighboring cells could not alter their fate. To follow cell mixing during Xenopus development, fluorescent dextran lineage tracers were microinjected into identified blastomeres at the 16-cell stage. The labelled descendants of the injected blastomeres were followed over several stages of embryogenesis. After gastrulation, the labelled descendants formed relatively coherent groups in characteristic regions of the embryo. By larval stages, most of the labelled descendants were still located in characteristic regions. However, coherence was less pronounced and individual descendants were located in many regions of the embryo. Hence, cell mixing is a slow, but progressive, process throughout Xenopus development. This is in sharp contrast to the extensive mixing that occurs during the early development of other vertebrates, such as zebrafish and mice. The slow cell mixing in Xenopus development suggests a simple mechanism for the consistent fates of cleavage-stage blastomeres. The stereotyped cell movements of embryogenesis redistribute the largely coherent descendants to characteristic locations in the embryo. The small amount of mixing that does occur would result in variable locations of a small proportion of the descendants; this could contribute to the observed variability of the blastomere fate map. Because cell mixing during Xenopus development is insufficient to challenge possible lineage restrictions, additional experiments must be performed to establish when and if lineage restrictions occur.     

Analysis of cell lineage in two- and four-cell mouse embryos

Compared with other animals, the embryos of mammals are considered to have a highly regulative mode of development. However, recent studies have provided a strong correlation between the first cleavage plane and the future axis of the blastocyst, but it is still unclear how the early axes of the preimplantation embryo reflect the future body axes that emerge after implantation. We have carried out lineage tracing during mouse embryogenesis using the Cre-loxP system, which allowed us to analyze cell fates over a long period of development. We used a transgenic mouse strain, CAG-CAT-Z as a reporter line. The descendants of the manipulated blastomere heritably express beta-galactosidase. We examined the distribution of descendants of a single blastomere in the 8.5-day embryo after labeling at the two-cell and four-cell stages. The derivatives of one blastomere in the two-cell embryo randomly mix with cells originating from the second blastomere in all cell layers examined. Thus we find cells from different blastomeres intermingled and localized randomly along the body axis. The results of labeling experiments performed in the four-cell stage embryo fall into three categories. In the first, the labeled cells were intermingled with non-labeled cells in a manner similar to that seen after labeling at the two-cell stage. In the second, labeled cells were distributed only in the extra-embryonic ectoderm layers. Finally in the third category, labeled cells were seen only in the embryo proper and the extra-embryonic mesoderm. Manipulated embryos analyzed at the blastocyst stage showed localized distribution of the descendants of a single blastomere. These results suggest that incoherent clonal growth and drastic cell mixing occurs in the early mouse embryo after the blastocyst stage. The first cell specification event, i.e., partitioning cell fate between the inner cell mass and trophectoderm, can occur between the two-cell and four-cell stage, yet the cell fate is not determined.     

FGF-mediated induction of ciliary body tissue in the chick eye

Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with the lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.     

A posterior centre establishes and maintains polarity of the Caenorhabditis elegans embryo by a Wnt-dependent relay mechanism

Cellular polarity is a general feature of animal development. However, the mechanisms that establish and maintain polarity in a field of cells or even in the whole embryo remain elusive. Here we provide evidence that in the Caenorhabditis elegans embryo, the descendants of P₁, the posterior blastomere of the 2-cell stage, constitute a polarising centre that orients the cell divisions of most of the embryo. This polarisation depends on a MOM-2/Wnt signal originating from the P₁ descendants. Furthermore, we show that the MOM-2/Wnt signal is transduced from cell to cell by a relay mechanism. Our findings suggest how polarity is first established and then maintained in a field of cells. According to this model, the relay mechanism constantly orients the polarity of all cells towards the polarising centre, thus organising the whole embryo. This model may also apply to other systems such as Drosophila and vertebrates.     

A Posterior Centre Establishes and Maintains Polarity of the Caenorhabditis elegans Embryo by a Wnt-Dependent Relay Mechanism

Cellular polarity is a general feature of animal development. However, the mechanisms that establish and maintain polarity in a field of cells or even in the whole embryo remain elusive. Here we provide evidence that in the Caenorhabditis elegans embryo, the descendants of P₁, the posterior blastomere of the 2-cell stage, constitute a polarising centre that orients the cell divisions of most of the embryo. This polarisation depends on a MOM-2/Wnt signal originating from the P₁ descendants. Furthermore, we show that the MOM-2/Wnt signal is transduced from cell to cell by a relay mechanism. Our findings suggest how polarity is first established and then maintained in a field of cells. According to this model, the relay mechanism constantly orients the polarity of all cells towards the polarising centre, thus organising the whole embryo. This model may also apply to other systems such as Drosophila and vertebrates.     

Development of the mesendoderm in the dendrobranchiate shrimp Sicyonia ingentis

Dendrobranchiate shrimp embryos form a 4-cell stage that resembles spiralians in its cell contacts, but cleavage proceeds radially without any further evidence of spiralian character. The fates of Sicyonia ingentis mesendoblasts were followed by nuclear staining and confocal microscopy. The dorsal mesendoblast produced yolk-endoderm, which proliferated from the anterior of the embryo to cover the dorsal interior. The ventral mesendoblast divided into the primordial endoblast and a cell that further divided into the primordial mesoteloblast and the primordial germ cell. The primordial endoblast divided into left and right endoblasts, which then underwent two teloblastic divisions, leaving behind two pairs of smaller descendants and the larger endoblasts at the dorsal posterior. The endoblasts then paused in cell division while extensive morphogenesis occurred in the ectoderm to form the naupliar segments. Ectoteloblasts formed at the posterior. The primordial mesoteloblast underwent two asymmetric divisions, synchronously with the endoblasts, to form two small descendants and M2 at the ventral posterior. From 15 to 18h, M2 divided laterally then dorsal-ventrally to form four descendants. The results extend the cell lineage of S. ingentis from the egg to the nauplius larva, and demonstrate that endoderm forms dorsal to teloblastic mesoderm from an early stage.     

Clonal and molecular analysis of the prospective anterior neural boundary in the mouse embryo

In the mouse embryo the anterior ectoderm undergoes extensive growth and morphogenesis to form the forebrain and cephalic non-neural ectoderm. We traced descendants of single ectoderm cells to study cell fate choice and cell behaviour at late gastrulation. In addition, we provide a comprehensive spatiotemporal atlas of anterior gene expression at stages crucial for anterior ectoderm regionalisation and neural plate formation. Our results show that, at late gastrulation stage, expression patterns of anterior ectoderm genes overlap significantly and correlate with areas of distinct prospective fates but do not define lineages. The fate map delineates a rostral limit to forebrain contribution. However, no early subdivision of the presumptive forebrain territory can be detected. Lineage analysis at single-cell resolution revealed that precursors of the anterior neural ridge (ANR), a signalling centre involved in forebrain development and patterning, are clonally related to neural ectoderm. The prospective ANR and the forebrain neuroectoderm arise from cells scattered within the same broad area of anterior ectoderm. This study establishes that although the segregation between non-neural and neural precursors in the anterior midline ectoderm is not complete at late gastrulation stage, this tissue already harbours elements of regionalisation that prefigure the later organisation of the head.     

Specification of secondary mesenchyme-derived cells in relation to the dorso-ventral axis in sea urchin blastulae

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg2-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg2 descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.     

Persistent expression of morphological abnormalities in the distant progeny of irradiated cells

The phenomenon of delayed heritable lethal damage (often referred to as ``lethal mutations') in the progeny of cells which survive irradiation is now well established, but little is known of the mechanism by which this cell death occurs. Current theories suggest a generalised genomic instability affecting all cells which leads to the production of some mutations which are lethal, or alternatively that a lethal mutation gene is activated, mutated or induced by radiation and leads to persistent and random cell death at high levels in the progeny. The aim of this study was to look at the morphology of progeny of irradiated cells at various times after irradiation to establish how widespread morphological abnormalities were in the population and whether there was any evidence that such abnormalities were clonal. Using two different cell lines, the results showed that morphological evidence possibly suggestive of apoptosis occurred in the cultures after all doses of radiation and up to 45 cell doublings after exposure. There was no evidence of a decrease in the numbers of damaged or dead cells in colonies with number of divisions after irradiation, or with decreasing original radiation dose. There was a significant dose-dependent increase in the number of cells with microvilli for both cell lines. The dose-dependency of this effect did not change with number of divisions after irradiation. It is clear that morphological evidence of cellular damage persists for several generations after the initial exposure. The effects are widespread in the cell population, and their constancy over time argues strongly for a general instability and against a clonal mechanism, since clonal descendants should die out and leave undamaged survivors. The lack of evidence for necrosis or senescence together with many morphological changes in the cultures suggestive of apoptosis could indicate an active mechanism of cell death. It is concluded that survivor populations of irradiated cells from two widely different mammalian cell lines demonstrate an altered phenotype including gross morphological changes. These result in a higher probability that cell division will fail to yield two healthy progeny. Received: 22 January 1996 / Accepted in revised form: 24 September 1996     

Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.     

Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

Summary Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division. divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all.     

Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate

Neurogenesis is widespread in the zebrafish adult brain through the maintenance of active germinal niches. To characterize which progenitor properties correlate with this extensive neurogenic potential, we set up a method that allows progenitor cell transduction and tracing in the adult zebrafish brain using GFP-encoding retro- and lentiviruses. The telencephalic germinal zone of the zebrafish comprises quiescent radial glial progenitors and actively dividing neuroblasts. Making use of the power of clonal viral vector-based analysis, we demonstrate that these progenitors follow different division modes and fates: neuroblasts primarily undergo a limited amplification phase followed by symmetric neurogenic divisions; by contrast, radial glia are capable at the single cell level of both self-renewing and generating different cell types, and hence exhibit bona fide neural stem cell (NSC) properties in vivo. We also show that radial glial cells predominantly undergo symmetric gliogenic divisions, which amplify this NSC pool and may account for its long-lasting maintenance. We further demonstrate that blocking Notch signaling results in a significant increase in proliferating cells and in the numbers of clones, but does not affect clone composition, demonstrating that Notch primarily controls proliferation rather than cell fate. Finally, through long-term tracing, we illustrate the functional integration of newborn neurons in forebrain adult circuitries. These results characterize fundamental aspects of adult progenitor cells and neurogenesis, and open the way to using virus-based technologies for stable genetic manipulations and clonal analyses in the zebrafish adult brain.     

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.     

Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish

In order to study lens-retina relationships during development, we cloned the zebrafish alphaA-crystallin cDNA and its promoter region. Using a 2.8-kb fragment of the zebrafish alphaA-crystallin promoter (z(alpha)Acry), we expressed the diphtheria toxin A fragment (DTA) in zebrafish embryos in a lens-specific manner. Injection of the z(alpha)Acry-DTA plasmid into eggs at the one-or two-cell stage resulted in the formation of small eyes, in which both lens and retina were reduced in size. In the DTA-expressing lenses, their fiber structure was disorganized, indicating that normal lens development had been abrogated. The neural retina also showed abnormal development, although this tissue did not express DTA. Lamination in the retina did not develop well, and molecular markers for the outer and inner plexiform layers were either abnormally expressed or absent. However, cell type-specific markers of ganglion and bipolar cells, as well as photoreceptors, were expressed in appropriate positions, indicating that initial differentiation of these retinal subpopulations occurred in the DTA-expressing embryos. Cell proliferation also proceeded normally in these embryos, although apoptosis was enhanced. These results suggest that the differentiated lens plays a critical role in the morphogenetic organization of retinal cells during eye development in zebrafish embryos.