Survival of xenogeneic grafts of embryonic pigment and carapace rudiments in embryos of Chelydra serpentina

In a study of survival of embryonic grafts in turtles, Chelydra was used as host and Chrysemys and Amyda as donors. Somites and overlying ectoderm with or without adjacent neural tube were transplanted. The operations were unilateral and orthotopic. The involved the anterior portion of the carapace. In other experiments, bilateral neural crest and dorsal neural tube were transplanted orthotopically. In experiments with Chrysemys as donor, pigment cells formed conspicuous red areas ventrally when neural crest was included in the graft. This pigment faded gradually but persisted for three or four years. When somites and adjacent ectoderm of Chrysemys carapace were transplanted, the graft area was lightly pigmented at hatching. This pigmentation increased subsequently. The Chrysemys grafts were either accepted or partially rejected. In cases of apparent complete acceptance, the graft region took on characteristics of the host. When Amyda served as donor of carapace rudiments, the graft area retained characteristics of the donor. At hatching, dark spots on a yellow background were present and scutes were absent. A few months after hatching, the graft area became necrotic. Subsequently, scutes with host characteristics or skin covered the graft area.     

Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus

Dorsal or ventral blastomeres of the 16- and 32-cell stage animal hemisphere were labeled with a lineage dye and transplanted into the position of a ventral, vegetal midline blastomere. The donor blastomeres normally give rise to substantial amounts of head structures and central nervous system, whereas the blastomere which they replaced normally gives rise to trunk mesoderm and endoderm. The clones derived from the transplanted ventral blastomeres were found in tissues appropriate for their new position, whereas those derived from the transplanted dorsal blastomeres were found in tissues appropriate for their original position. The transplanted dorsal clones usually migrated into the host's primary axis (D1.1, 92%; D1.1.1, 69%; D1.1.2, 100%), and in many cases they also induced and populated a secondary axis (D1.1, 43%; D1.1.1, 67%; D1.1.2, 63%). Bilateral deletion of the dorsal blastomeres resulted in partial deficits of dorsal axial structures in the majority of cases, whereas deletions of ventral midline blastomeres did not. When the dorsal blastomeres were cultured as explants they elongated. Notochord and cement glands frequently differentiated in these explants. These studies show that the progeny of the dorsal, midline, animal blastomeres: (1) follow their normal lineage program to populate dorsal axial structures after the blastomere is transplanted to the opposite pole of the embryo; (2) induce and contribute to a secondary axis from their transplanted position in many embryos; (3) are important for the normal formation of the entire length of the dorsal axis; and (4) autonomously differentiate in the absence of exogenous growth factor signals. These data indicate that by the 16-cell stage, these blastomeres have received instructions regarding their fate, and they are intrinsically capable of carrying out some of their developmental program.     

Demonstration of neural induction using nuclear markers inXenopus

Summary Two nuclear markers were used to investigate the origin of cells in secondary embryos ofXenopus induced by dorsal lip transplants, and to determine the ability of the chordomesoderm to direct cells to change their fates.³H-thymidine was used to label cells transplanted between individualX. laevis embryos, and nuclear quinacrine fluorescence was used to distinguishX. borealis tissues transplanted toX. laevis hosts. In the first set of experiments, dorsal lip tissue (also known as the dorsal marginal zone; DMZ) was transplanted to the ventral marginal zone (VMZ) of host embryos. The marginal zone is the toroid of presumptive mesodermal cells which involutes during gastrulation. Examination of the secondary embryos resulting from these grafts revealed that their notochords were derived almost exclusively from transplanted cells whereas their nervous systems and somites were composed almost entirely of host cells. Next, the nuclear markers were used to show the normal fates of the tissue of the ventral equatorial region immediately above the VMZ by orthotopic grafting. This tissue was found to give rise to structures in the ventral posterior portions of the tailbud embryo. Finally, the same ventral tissue was labeled and transplanted to the dorsal equatorial region above the DMZ. As a result, it was induced to change its fate and become neural. These results lend unequivocal support to Spemann's theory of neural induction which has recently been questioned.     

Artificially Applied Tensions Normalize Development of Relaxed Xenopus laevisEmbryos

Relaxation of tensions of the surface of Xenopus laevisembryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by the incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.     

Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm

The successful organization of the vertebrate body requires that local information in the embryo be translated into a functional, global pattern. Somite cells form the bulk of the musculoskeletal system. Heterotopic transplants of segmental plate along the axis from quail to chick were performed to test the correlation between autonomous morphological patterning and Hox gene expression in somite subpopulations. The data presented strengthen the correlation of Hox gene expression with axial specification and focus on the significance of Hox genes in specific derivatives of the somites. We have defined two anatomical compartments of the body based on the embryonic origin of the cells making up contributing structures: the dorsal compartment, formed from purely somitic cell populations; and the ventral compartment comprising cells from somites and lateral plate. The boundary between these anatomical compartments is termed the somitic frontier. Somitic tissue transplanted between axial levels retains both original Hox expression and morphological identity in the dorsal compartment. In contrast, migrating lateral somitic cells crossing the somitic frontier do not maintain donor Hox expression but apparently adopt the Hox expression of the lateral plate and participate in the morphology appropriate to the host level. Dorsal and ventral compartments, as defined here, have relevance for experimental manipulations that influence somite cell behavior. The correlation of Hox expression profiles and patterning behavior of cells in these two compartments supports the hypothesis of independent Hox codes in paraxial and lateral plate mesoderm.     

Two essential processes in the formation of a dorsal axis during gastrulation ofCynops embryo

The isolated upper marginal zone from the initial stage ofCynops gastrulation is not yet determined to form the dorsal axis mesoderm: notochord and muscle. In this experiment, we will indicate where the dorsal mesoderm-inducing activity is localized in the very early gastrula, and what is an important event for specification of the dorsal axis mesoderm during gastrulation. Recombination experiments showed that dorsal mesoderm-inducing activity was localized definitively in the endodermal epithelium (EE) of the lower marginal zone, with a dorso-ventral gradient; and the EE itself differentiated into endodermal tissues, mainly pharyngeal endoderm. Nevertheless, when dorsal EE alone was transplanted into the ventral region, a secondary axis with dorsal mesoderm was barely formed. However, when dorsal EE was transplanted with the bottle cells which by themselves were incapable of mesoderm induction, a second axis with well-developed dorsal mesoderm was observed. When the animal half with the lower marginal zone was rotated 180° and recombined with the vegetal half, most of the rotated embryos formed only one dorsal axis at the primary blastopore side. The present results suggest that there are at least two essential processes in dorsal axis formation: mesoderm induction of the upper marginal zone by endodermal epithelium of the lower marginal zone, and dorsalization of the upper dorsal marginal zone evoked during involution.     

Engraftment and differentiation of neocortical progenitor cells transplanted to the embryonic brain in utero

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ß-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.     

Prospective Neural Areas and their Morphogenetic Movements during Neural Plate Formation in the Xenopus Embryo. II. Disposition of Transplanted Ectoderm Pieces of X. borealis Animal Cap in Prospective Neural Areas of Albino X. laevis gastrulae.

Small pieces of the animal cap of X. borealis gastrulae were transplanted into various regions of the noninvoluting marginal zone of albino X. laevis gastrulae, and the distribution of the donor cells was analyzed by quinacrine fluorescence staining.
The present study indicated that the prospective central nervous system (CNS) lies as a belt-shaped area in the noninvoluting marginal zone of early gastrulae. This belt-shaped prospective neural area extends as far as 0.7 mm (115° to the vegetal pole) above the blastopore in the dorsal midline and 1.3 mm lateral (130° to the dorsal midline) to the dorsal midline. The ectoderm of the dorsal region extends in the animal-vegetal direction and forms the ventral side of the CNS. The dorsalateral and lateral regions converge toward the dorsal midline and extended in the animal-vegetal direction. The former constitutes the lateral side of the anterior CNS, and the latter the dorso-lateral side of the posterior CNS.
The outer layer of ectoderm which was transplanted onto the inner layer of the host gastrula differentiated into neural tissues.
The prospective areas of the CNS and their morphogenetic movement during Xenopus embryogenesis are also discussed with regard to neural induction.     

Early cellular interactions promote embryonic axis formation in Xenopus laevis

We have attempted to define the location and mode of action of axial determinants in the egg of Xenopus laevis. To this end, we transplanted small numbers of blastomeres from normal 64-cell stage embryos into synchronous recipient embryos which had been irradiated with ultraviolet light prior to first cleavage. Without transplantation, such embryos fail to develop dorsal structures of the embryonic body axis. We found that one to three blastomeres transplanted from the vegetal-most octet of cells can effect complete or partial rescue of of axis development in a recipient, provided that the donor cells derive from the quadrant just under the prospective dorsal marginal region. These same cells, when transplanted into the ventral vegetal quadrant of a normal 64-cell embryo, cause the formation of a complete second body axis. In contrast, other cells from the vegetal octet of normal donors fail to cause axis formation. When the rescuing donor cells are labeled with a lineage-restricted fluorescent marker, we find that their progeny do not contribute to the axial structures of the recipient. Progeny of the transplanted cells are found below the level of the blastopore in the early gastrula and eventually give rise to portions of the gut, as is their fate in normal development. These results, in agreement with those of Nieuwkoop (P.D. Nieuwkoop, 1977, Curr. Top. Dev. Biol. 11, 115-132), imply that the dorsal-most vegetal cells of the 64-cell embryo receive from the egg cytoplasm a set of determinants enabling them to induce neighboring cells to undertake axis formation. We discuss the relationship between axis induction in rescued irradiated embryos and axis determining processes in normal embryogenesis.     

Capacity of neural crest cells from various axial levels to participate in thymic development

Summary In order to assess the capacity of neural crest from different sources to participate in thymic development, neural crest from selected axial levels was transplanted unilaterally from quail donors to the region in chick hosts from which neural crest cells normaly migrate to interact with the primordial thymus. The greatest representation of donor cells was observed after isotopic transplantation and when donor tissue was taken from the hyoid and mesencephalic regions of the neural crest. The capacity for transplants to contribute cells decreased both anteriorly and posteriorly, so that neural crest close to the usual origin of mesenchyme-producing cells contributed a larger number of donor cells around the developing thymus than neural crest from anterior and posterior regions. Cells from the transplant were inserted as an addition to the host chick cells. Thus, a special relationship and capacity for interaction in thymic development is expressed by neural crest at usual levels over a limited span of axial regions, but to some extent by all regions. This study has established that the capacity for neural crest cells from different axial levels to interact with developing organs is not uniform, but may vary, depending upon the nature of the interaction with a particular organ.This study was supported by Grant No. 2332, The Council for Tobacco Research, USA, Inc.     

Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos

Relaxation of tensions of the surface of Xenopus laevis embryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching, but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.     

Role of mechano-dependent cell movements in the establishment of spatial organization of axial rudiments in <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

Trajectories of individual cell movements and patterns of differentiation in the axial rudiments in suprablastoporal areas (SBA) in whole embryos of Xenopus laevis artificially stretched in the transverse direction up to 120–200% from the initial length at the early gastrula stage were mapped. We observed the impairment of anisotropic cell movements of longitudinal stretching and latero-medial convergence inherent for SBA. Axial rudiments occurred in all cases, but their location was completely impaired and dramatically different from the normal topology for moderate (120–140%) stretching. Stronger stretching caused a partial ordering of the whole axial complex and its reorientation toward stretching. We concluded that induction factors determine short-range order in their arrangement in SBA, whereas anisotropic cell movements in any direction are needed for long-range order. Moderate transverse stretching destroys normally oriented anisotropy, but it is not enough for establishment of the anisotropy oriented perpendicular to the normal. This explains the disorder at light stretching. The main conclusion of this study is that anisotropic tensions of embryonic tissues play role of long-range order parameters of cell differentiation.     

Relocations of cell convergence sites and formation of pharyngula-like shapes in mechanically relaxed <Emphasis Type="Italic">Xenopus</Emphasis> embryos

Influence of the relaxation of mechanical tensions upon collective cell movements, shape formation, and expression patterns of tissue-specific genes has been studied in Xenopus laevis embryos. We show that the local relaxation of tensile stresses within the suprablastoporal area (SBA) performed at the early-midgastrula stage leads to a complete arrest of normal convergent cell intercalation towards the dorsal midline. As a result, SBA either remains nondeformed or protrudes a strip of cells migrating ventralwards along one of the lateral lips of the opened blastopore. Already, few minutes later, the tissues in the ventral lip vicinity undergo abnormal transversal contraction/longitudinal extension resulting in the abnormal cell convergence toward ventral (rather than dorsal) embryo midline. Within a day, the dorsally relaxed embryos acquire pharyngula-like shapes and often possess tail-like protrusions. Their antero-posterior and dorso-ventral polarity, as well as expression patterns of pan-neural (Sox3), muscular cardiac actin, and forebrain (Otx2) genes substantially deviate from the normal ones. We suggest that normal gastrulation is permanently controlled by mechanical stresses within the blastopore circumference. The role of tissue tensions in regulating collective cell movements and creating pharyngula-like shapes are discussed.     

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.     

Inductive activity of the posterior tip of planula in the marine hydroid <Emphasis Type="Italic">Dynamena pumila</Emphasis>

Activity of organizer regions is required for body plan formation in the developing organism. Transplanting a fragment of such a region to a host organism leads to the formation of a secondary body axis that consists of both the donor’s and the host’s tissues (Gerhart, 2001). The subject of this study, the White Sea hydroid cnidarian Dynamena pumila L. (Thecaphora, Sertulariidae), forms morphologically advanced colonies in the course of complex metamorphosis of the planula larva. To reveal an organizer region, a series of experiments has been performed in which small fragments of donor planula tissues were transplanted to embryos at the early and late gastrula stage, as well as to planulae. Only transplantations of a posterior tip fragment of a donor planula to a host planula of the same age led, in the course of metamorphosis, to the formation of a secondary shoot, which involved up to 50% of the host’s tissues. After transplantations of tissue fragments of the anterior tip and the middle of the planula body, the formation of any ectopic structures was never observed. It was concluded that the posterior tip of the planula has organizer properties in Dynamena.     

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis

To study the regulation of the dorsal axial structures, we removed the right animal dorsal and the right vegetal dorsal cells from an 8-cell embryo of Xenopus laevis .
Most of the right dorsal cell-deficient embryos developed to normally proportioned tailbud embryos. No detectable delay was observed in their development. Examinations of serial sections revealed that they had restored bilateral symmetry. The cell numbers of the somite and the notochord had recovered to more than 90% and 70%, respectively, those of controls. Since the right dorsal cell-deficient embryo retained roughly three-quarters of the prospective region for the somites and half of that for the notochord, respectively, the cell number was more than that expected from the remaining prospective regions. Cell lineage analyses showed that progeny of the right ventral cells had formed almost all of the right dorsal axial structures, which are normally formed by the progeny of the right dorsal cells. However, almost all the notochord cells had been derived from the remaining left dorsal cells.
These results indicate that some quantitative aspects of regulation as expressed in terms of the cell number were different between the two tissues examined.     

Acquisition of developmental autonomy in the equatorial region of the Xenopus embryo

This paper describes a continuing effort to define the location and mode of action of morphogenetic determinants which direct the development of dorsal body axis structures in embryos of the frog Xenopus laevis. Earlier results demonstrated that presumptive endodermal cells in one vegetal quadrant of the 64-cell embryo can, under certain experimental conditions, induce partial or complete body axis formation by progeny of adjacent equatorial cells. (R.L. Gimlich and J.C. Gerhart, 1984, Dev. Biol. 104, 117-130). I have now assessed the importance of other blastomeres for embryonic axis formation in a series of transplantation experiments using cells from the equatorial level of the 32-cell embryo. The transplant recipients were embryos which had been irradiated with ultraviolet light before first cleavage. Without transplantation, embryos failed to develop the dorsal structures of the embryonic body axis. However, cells of these recipients were competent to respond to inductive signals from transplanted tissue and to participate in normal embryogenesis. Dorsal equatorial cells, but not their lateral or ventral counterparts, often caused partial or complete body axis development in irradiated recipients, and themselves formed much of the notochord and some prechordal and somitic mesoderm. These are the same structures that they would have formed in the normal donor. Thus, the dorsal equatorial blastomeres were often at least partially autonomous in developing according to their prospective fates. In addition, they induced progeny of neighboring host cells to contribute to the axial mesoderm and to form most of the central nervous system. The frequency with which such transplants caused complete axis formation in irradiated hosts increased when they were made at later and later cleavage stages. In contrast, the inductive activity of vegetal cells remained the same or declined during the cleavage period. These and other results suggest that the egg cytoplasmic region containing "axial determinants" is distributed to both endodermal and mesodermal precursors in the dorsal-most quadrant of the early blastula.     

Behavior of cells in artificially made cell aggregates and tissue fragments after grafting to developing hind limb buds in Xenopus laevis

During vertebrate limb development, the limb bud grows along the proximo-distal (P-D) direction, with the cells changing their adhesiveness. To know whether the position-related differences in cell adhesiveness are actually utilized by morphogenesis to constitute limb structures, we grafted cell aggregates made of dissociated cells derived from different positions and stages of developing hind limb buds into developing hind limb buds and observed the behavior of the cells. Cell aggregates made of dissociated mesenchymal cells from two different origins were implanted in different positions and stages of limb buds or grafted on limb stumps made by cutting. The two grafted cell populations in the aggregate always sorted out from each other, but their patterning of sorting-out was quite different according to the transplanted regions. In summary, cells in the aggregate that have closer positional identity to the transplanted site were always situated at the boundary between host and donor cells. The pattern of sorting-out seemed to be determined by the relative adhesiveness of surrounding cells to the constituent cells of the aggregates. We also transplanted fragments dissected out from different regions along the P-D axis into st. 50 limb buds. The descendants of grafted cells moved distally to the region corresponding to their positional identity and participated in the formation of more distal structures from that point. These results suggest that the difference in cell adhesiveness may probably play a role in arranging cells along the P-D axis of a developing limb bud.     

Formation and cellular structure of the lines of tension in the axial rudimenta of amphibian embryos]

The lines of mechanical tension (cross-lines) in axial rudiments of the amphibian embryo represent bands of polarized cells. They form in the inner layers of the rudiments as separate bundles of polarized cell which, then, merge, attain the external surface and gather in lengthy planes (cross-planes) and, later, degrade. The primary inductor induces the formation of cross-lines in the ventral ectoderm of the early gastrula. The growth of cross-lines in considered as one of the types of contact cells polarization. The morphogenetic role of contact polarization is discussed. The connection between the subsequent tension patterns is based on the fact that the lines of exit of the cross-planes on the surface of the embryo coincide with the direction of the previously established tensions.