Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.     

Cell motility driving mediolateral intercalation in explants of Xenopus laevis.

In Xenopus, convergence and extension are produced by active intercalation of the deep mesodermal cells between one another along the mediolateral axis (mediolateral cell intercalation), to form a narrower, longer array. The cell motility driving this intercalation is poorly understood. A companion paper shows that the endodermal epithelium organizes the outermost mesodermal cells immediately beneath it to undergo convergence and extension, and other evidence suggests that these deep cells are the most active participants in mediolateral intercalation (Shih, J. and Keller, R. (1992) Development 116, 887-899). In this paper, we shave off the deeper layers of mesodermal cells, which allows us to observe the protrusive activity of the mesodermal cells next to the organizing epithelium with high resolution video microscopy. These mesodermal cells divide in the early gastrula and show rapid, randomly directed protrusive activity. At the early midgastrula stage, they begin to express a characteristic sequence of behaviors, called mediolateral intercalation behavior (MIB): (1) large, stable, filiform and lamelliform protrusions form in the lateral and medial directions, thus making the cells bipolar; (2) these protrusions are applied directly to adjacent cell surfaces and exert traction on them, without contact inhibition; (3) as a result, the cells elongate and align parallel to the mediolateral axis and perpendicular to the axis of extension; (4) the elongate, aligned cells intercalate between one another along the mediolateral axis, thus producing a longer, narrower array. Explants of essentially a single layer of deep mesodermal cells, made at stage 10.5, converge and extend by mediolateral intercalation. Thus by stage 10.5 (early midgastrula), expression of MIB among deep mesodermal cells is physiologically and mechanically independent of the organizing influence of the endodermal epithelium, described previously (Shih, J. and Keller, R. (1992) Development 116 887-899), and is the fundamental cell motility underlying mediolateral intercalation and convergence and extension of the body axis.     

Induction of blood cells in Xenopus embryo explants

 A Xenopus-specific anti-leukocyte monoclonal antibody designated XL-2 was isolated and used to identify leukocytes in tailbud embryos and activin A-treated explants of blastula animal cap. XL-2 bound to a 135-kDa polypeptide in western blots of protein extracts from adult thymocytes, tailbud embryos, tadpoles, and explants. In cell suspensions, it immunostained the cell surface of all types of adult leukocytes including lymphocytes, monocyte/macrophages, thrombocytes, and granulocytes. At embryonic stage 24, immunocytochemistry revealed XL-2-positive leukocytes, the earliest time at which such cells have been recognized. Whole-mount staining of tailbud embryos and tadpoles showed a widely dispersed population of XL-2-reactive leukocytes, many of which had elongated shapes and ameboid pseudopodia. In activin A-treated animal caps, XL-2 recognized a subpopulation of cells within the lumen of the central fluid-filled cavity as well as cells in the interstitium of mesenchymal and mesothelial components of the explant. Together, activin A and human interleukin-11 induced 100% of explants to form lumenal blood cells. Compared to activin A alone, murine stem cell factor plus activin A significantly increased the numbers of XL-2-reactive leukocytes and erythrocytes. These results support the view that activin A induces leukocyte and erythrocyte progenitors during Xenopus embryogenesis. Received: 29 August 1997 / Accepted: 28 October 1997     

In vitro induction of the pronephric duct in Xenopus explants

The earliest form of embryonic kidney, the pronephros, consists of three components: glomus, tubule and duct. Treatment of the undifferentiated animal pole ectoderm of Xenopus laevis with activin A and retinoic acid (RA) induces formation of the pronephric tubule and glomus. In this study, the rate of induction of the pronephric duct, the third component of the pronephros, was investigated in animal caps treated with activin A and RA. Immunohistochemistry using pronephric duct-specific antibody 4A6 revealed that a high proportion of the treated explants contained 4A6-positive tubular structures. Electron microscopy showed that the tubules in the explants were similar to the pronephric ducts of normal larvae, and they also expressed Gremlin and c-ret, molecular markers for pronephric ducts. These results suggest that the treatment of Xenopus ectoderm with activin A and RA induces a high rate of differentiation of pronephric ducts, in addition to the differentiation of the pronephric tubule and glomus, and that this in vitro system can serve as a simple and effective model for analysis of the mechanism of pronephros differentiation.     

Glutamate induces apoptosis in cultured spiral ganglion explants

Traumatic sound exposure, aminoglycoside antibiotics, cochlea ischemia or traumatic stress leads to an excessive release of glutamate from inner hair cells into the synaptic cleft. The high glutamate concentration can cause a swelling and destruction of the dendrites of spiral ganglion neurons of type I as well as a reduction in the number of neurons. This may be a cause of hearing loss. The mechanism causing the reduction of neurons is still not known. Apoptosis, also called programmed cell death, could be involved. In this study, cultured spiral ganglion explants were incubated with glutamate in high concentrations. Neurite outgrowth was determined and additionally a new method was established for studying the morphology of single spiral ganglion neurons. For the first time it was shown that glutamate induces apoptosis of spiral ganglion neurons, which could be blocked selectively by a caspase-3 inhibitor. This could offer a new therapeutic strategy for hearing disorders.     

Ciliary epithelia of the mammalian eye in cultured explants

The ultrastructural characteristics of ciliary epithelium from bovine, pigmented rabbit, and fetal albino rabbit were studied in cultured explants. The tips of ciliary processes were cultured in plastic dishes with Dulbecco Modified Eagle Medium (DMEM) containing 5% fetal bovine serum. More than half of the explants adhered to the plastic culture dish, and epithelial cells spread as monolayers within a few days. Initially the explant contains two layers, the outer (nonpigmented cells) and the inner (pigmented cells). Later the explant exhibits three layers: 1) outermost lightly pigmented flattened cells, 2) an outer layer of non-pigmented cells, and 3) an inner layer of densely pigmented cuboidal cells. The cells of the outermost layer are continuous with the cells of the inner layer. A narrow space lies between the outermost layer and the outer layer. The columnar cells in the outer layer contain well developed organelles but no pigment granules; they possess a basement membrane, lateral interdigitations, and junctional complexes near their apices. Numerous focal junctions and some ciliary channel-like structures were detected between the columnar cells of the outer layer and the cuboidal cells of the inner layer. The cuboidal cells of the inner layer are filled with pigment granules. These observations suggest that the columnar cells of the outer layer are nonpigmented epithelium, the cuboidal cells of the inner layer are pigmented epithelium, and the flattened cells in the outermost layer are derived from pigmented epithelium.     

Biochemical changes in cultured foetal rat liver explants.

Liver explants from 20-day-old foetuses cultured for 48h in the absence of serum released 70% of their total soluble protein content into the medium. In the presence of serum this loss still amounted to 60%. The concentration of total particulate protein remained unchanged but there was some translocation of mitochondrial enzymes to the cytosol, and enzymes expected to increase during this stage of development failed to do so. The addition of cortisol plus glucagon (to serum-containing media) did not decrease the loss of total soluble protein from the explants but induced considerable tyrosine aminotransferase activity which was not released into the medium. The observations suggest that under the usual culture conditions a minority of the cells retain their functional integrity. The extent of deterioration, not reflected in histologically visible necrosis or cell damage, can be conveniently monitored by the malate dehydrogenase activity released to the medium.     

Retinal ganglion cell differentiation in cultured mouse retinal explants

The availability of genetically engineered mice harboring specific mutations in genes affecting one or more retinal cell types affords new opportunities for investigating the genetic regulatory mechanisms of vertebrate retina formation. When identifying critical regulatory genes involved in retina development it is often advantageous to complement in vivo analysis with in vitro characterization. In particular, by combining classical techniques of retinal explant culturing with gene transfer procedures relying on herpes simple virus (HSV) amplicon vectors, gain-of-function analysis with genes of interest can be performed quickly and efficiently. Here, details are provided for isolating and culturing explants containing retinal progenitor cells and for infecting the explants with HSV expression vectors that perturb or rescue retinal ganglion cells, the first cell type to differentiate in the retina. In addition, the availability of sensitive techniques to monitor gene expression, including detection of reporter gene expression using antibodies and detection of endogenous marker gene expression using quantitative RT-PCR, provides an effective means for comparing wild-type and mutant retinas from genetically engineered mice.     

Organization and rearrangement of immunoglobulin M genes in the amphibian Xenopus.

Sequences of immunoglobulin (Ig) cDNA clones of Xenopus laevis show that at least three different VH families are expressed in association with different JH elements and different isotypes of Ig constant regions. In genomic Southern blot analysis, the VH probes for each family hybridize to a distinct set of multiple DNA fragments. In contrast, the genomic JH elements and the IgM constant region gene are localized in a single DNA fragment of approximately 15 kb. Genomic VH elements contain regulatory sequences similar to those in VH genes of shark, fish and mammals and have a leader peptide sequence that contains an intron; they encode the VH region until residue 95 and have heptamer--23-bp--nonamer motifs similar to the rearrangement signal sequences (RSS) in all other vertebrate VH elements. The six genomic JH elements so far sequenced have a nonamer--23-bp--heptamer motif at their 5' end. These RSS motifs imply the existence of DH elements. The comparison of cDNA clones that contain similar constant regions but different VH regions or JH elements suggest rearrangement events. This is shown by Southern blot analysis of erythrocyte and B cell DNA with a JH probe. Thus, the overall organization of the Xenopus Ig gene locus is similar to that of mammals but strikingly different from shark.     

The Xenopus Cell Cycle: An Overview

Oocytes, eggs and embryos from the frog Xenopus laevis have been an important model system for studying cell-cycle regulation for several decades. First, progression through meiosis in the oocyte has been extensively investigated. Oocyte maturation has been shown to involve complex networks of signal transduction pathways, culminating in the cyclic activation and inactivation of Maturation Promoting Factor (MPF), composed of cyclin B and cdc2. After fertilisation, the early embryo undergoes rapid simplified cell cycles which have been recapitulated in cell-free extracts of Xenopus eggs. Experimental manipulation of these extracts has given a wealth of biochemical information about the cell cycle, particularly concerning DNA replication and mitosis. Finally, cells of older embryos adopt a more somatic-type cell cycle and have been used to study the balance between cell cycle and differentiation during development.     

Cell lineage and segmentation in the leech

Segments in the leech arise by the proliferation of longitudinally arrayed bandlets of blast cells derived from ten identifiable embryonic stem cells, two M, two N, four O/P and two Q teloblasts. In each bandlet, older blast cells lie ahead of those born later. By using microinjected cell lineage tracers it was shown previously that the teloblasts give rise to characteristic cell patterns made up of segmentally iterated complements of progeny designated as M, N, O, P and Q kinship groups. When a teloblast is injected after it has begun generating blast cells, a boundary is observed later in development between anterior, unlabelled progeny of blast cells produced before injection and posterior, labelled progeny of blast cells produced after injection. We have examined such boundaries in detail to establish the precise relationship between blast cell clones and segments, with the following conclusions: (i) in the M, O and P cell lines, one blast cell generates one segmental complement of progeny, but serially homologous blast clones intermix so that no segment boundaries can be defined based on primary blast cell clones; (ii) in the N and Q cell lines, two blast cells are required to generate a complete segmental complement of progeny; (iii) in the process of forming the germinal plate, cells derived from the N and Q teloblasts move past those derived from the M and O/P teloblasts, so that consegmental blast cell clones do not come into register until well after the establishment of segmentally iterated units within each bandlet.     

Comparison of tuber and shoot formation from in vitro cultured potato explants

The development of axillary buds of potato (Solanum tuberosum L.) plants, cultured in vitro, was analyzed. Depending on the composition of the culture medium, the buds developed into either tubers (medium with 8% sucrose), shoots (1% sucrose), or stolons (8% sucrose and 0.5 μM gibberellin). Endogenous sugar and starch levels, and key-enzymes involved in the conversion of sucrose to starch were determined at different stages of development. Moreover, the spatial distribution of sugar levels and enzyme activities were determined within the developing structures. Glucose and fructose decreased upon tuber formation, most noticeably in the swelling parts, where also starch accumulated. The activities of sucrose synthase, fructokinase and ADP-glucose pyrophosphorylase were highest under tuber-inducing conditions, the increase being confined to the tubers, and absent in the subtending stolons. It is concluded that changes in the measured parameters, observed under tuberizing conditions, are specifically related to the formation of the tuber, and are confined to the swelling part only. This revised version was published online in June 2006 with corrections to the Cover Date.