Regulated expression of the retinoblastoma gene product by fibroblast growth factor but not by activin during mesoderm induction in Xenopus

 The retinoblastoma (RB) gene is a tumor suppressor gene that plays an important role in cell cycle arrest and in the terminal differentiation of skeletal myoblasts. Differentiation into muscle occurs in Xenopus embryo explants during mesoderm induction by fibroblast growth factor (FGF) or activin A. We examined expression of the RB gene product (pRB) during mesoderm induction in vivo and in vitro. We show that hypo- and hyper-phosphorylated forms of pRB are present during early development and that expression of both forms increases significantly during the blastula stage, concomitant with mesoderm induction. Further investigation revealed that pRB is enriched in the presumptive mesoderm of the blastula stage embryo. In animal cap explants induced by Xenopus bFGF (XbFGF), pRB expression levels increased approximately tenfold while no increase was observed in explants induced by activin. However, when explants were induced by XbFGF in the presence of sodium orthovanadate, a compound previously shown to synergize with FGF to produce more dorsal ”activin-like” inductions than FGF alone, only a slight increase in pRB expression was observed. Furthermore, upregulation of pRB during mesoderm induction in vitro displayed an inverse correlation with expression of XFKH1, a marker for notochord. These results suggest that pRB may be important for patterning along the dorsoventral axis. Received: 22 February 1996 / Accepted: 20 September 1996     

Over-expression of fibroblast growth factors in Xenopus embryos.

A number of forms of fibroblast growth factor (FGF) were over-expressed within Xenopus embryos by injection of synthetic FGF mRNAs into fertilized eggs. Injected embryos showed abnormalities in development which were mainly secondary to a disruption of gastrulation movements. The effects observed after injection of bFGF mRNA, however, were much less severe than those observed after injection of an altered form of bFGF mRNA which differs only by the addition of a signal sequence for secretion, or of another member of the FGF family, kFGF, which is normally efficiently secreted. All forms of FGF caused the induction of mesoderm in animal cap explants isolated from blastulae, but the amount of bFGF mRNA required to induce the formation of significant levels of mesoderm was higher by a factor of over a hundred than that of the FGFs which contain a signal sequence for secretion. Over-expressed bFGF accumulated in the nuclei of blastulae but did not necessarily cause mesoderm formation. These results show that FGFs must be secreted from the cells in which they are synthesised in order to act efficiently as mesoderm inducing factors and suggest that bFGF itself, which does not contain a signal sequence for secretion, is unlikely to be directly involved in mesoderm induction during early embryonic development.     

Well-defined growth factors promote cardiac development in axolotl mesodermal explants

The effect of growth factors on the formation of cardiac mesoderm in the urodele, Ambystoma mexicanum (axolotl), has been examined using an in vitro explant system. It has previously been shown that cardiac mesoderm is induced by pharyngeal endoderm during neurula stages in urodeles. In this study, explants of prospective cardiac mesoderm from early neurula stage embryos rarely formed beating cardiac tissue in culture. When transforming growth factor beta-1 (TGF-beta 1) or platelet-derived growth factor BB (PDGF) was added to such explants, the frequency of heart tissue formation increased markedly. The addition of other growth factors to these explants did not enhance cardiac mesoderm formation. The addition of basic fibroblast growth factor (bFGF) to prospective heart mesoderm derived from later stage embryos resulted in a decreased tendency to form cardiac tissue. These results suggest that growth factors analogous to TGF-beta 1, PDGF, and bFGF may regulate the initial stages of vertebrate cardiac development in vivo.     

Acidic fibroblast growth factor in the developing rat embryo

Compared to basic fibroblast growth factor (bFGF), a widely distributed, broad spectrum mitogen and mesoderm inducer, acidic fibroblast growth factor (aFGF) is reported to have an essentially neural distribution and to be undetectable in the early embryo. In the present investigation, we used immunoblotting and immunochemistry to assess the cellular and tissue distributions of aFGF and bFGF in 11-20-d rat embryos. Immunoblotting of crude and heparin-bound embryo extracts revealed faint bands at the expected 17-18-kD and predominant bands at an apparent molecular mass of 26 to 28-kD (despite reducing conditions) using multiple specific antibodies for aFGF and bFGF. Pretreatment with 8 M urea yielded 18-20-kD aFGF and bFGF and some 24-26-kD bFGF. Immunoreactivity for both aFGF and bFGF was positive and similar in the cytoplasm, nuclei, and extracellular matrix of cells of neuroectodermal and mesodermal origin, while it was negative in endoderm-derived cells. The distribution of immunoreactive aFGF and bFGF also showed changes during development that were associated with the process of cellular and tissue differentiation. For example, intensity and extent of immunoreactivity for both peptides progressively increased in the middle layer of the spinal cord with increasing differentiation of the neural cells. The immunostaining patterns were very similar for aFGF and bFGF for each organ and at each stage. In conclusion, high molecular mass forms of immunoreactive aFGF and bFGF are present in the rat embryo. Acidic FGF and bFGF are both widely distributed in tissues of neuroectodermal and mesodermal origin, and their distribution was very similar.     

The role of growth factors in embryonic induction in Xenopus laevis.

Establishment of the body pattern in all animals, and especially in vertebrate embryos, depends on cell interactions. During the cleavage and blastula stages in amphibians, signal(s) from the vegetal region induce the equatorial region to become mesoderm. Two types of peptide growth factors have been shown by explant culture experiments to be active in mesoderm induction. First, there are several isoforms of fibroblast growth factor (FGF), including aFGF, bFGF, and hst/kFGF. FGF induces ventral, but not the most dorsal, levels of mesodermal tissue; bFGF and its mRNA, and an FGF receptor and its mRNA, are present in the embryo. Thus, FGF probably has a role in mesoderm induction, but is unlikely to be the sole inducing agent in vivo. Second, members of the transforming growth factor-beta (TGF-beta) family. TGF-beta 2 and TGF-beta 3 are active in induction, but the most powerful inducing factors are the distant relatives of TGF-beta named activin A and activin B, which are capable of inducing all types of mesoderm. An important question relates to the establishment of polarity during the induction of mesoderm. While all regions of the animal hemisphere of frog embryos are competent to respond to activins by mesoderm differentiation, only explants that include cells close to the equator form structures with some organization along dorsoventral and anteroposterior axes. These observations suggest that cells in the blastula animal hemisphere are already polarized to some extent, although inducers are required to make this polarity explicit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.     

Expression of syndecan, a putative low affinity fibroblast growth factor receptor, in the early mouse embryo.

Syndecan is an integral membrane proteoglycan that binds cells to several interstitial extracellular matrix components and binds to basic fibroblast-growth factor (bFGF) thus promoting bFGF association with its high-affinity receptor. We find that syndecan expression undergoes striking spatial and temporal changes during the period from the early cleavage through the late gastrula stages in the mouse embryo. Syndecan is detected initially at the 4-cell stage. Between the 4-cell and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but is absent from regions of cell-cell contact. At the blastocyst stage, syndecan is first detected at cell-cell boundaries throughout the embryo and then, at the time of endoderm segregation, becomes restricted to the first site of matrix accumulation within the embryo, the interface between the primitive ectoderm and primitive endoderm. During gastrulation, syndecan is distributed uniformly on the basolateral cell surfaces of the embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoderm cells, suggesting that it contributes to the process of mesoderm specification. In the extraembryonic region, syndecan is not detectable on most cells of the central core of the ectoplacental cone, but is strongly expressed by cells undergoing trophoblast giant cell differentiation and remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core protein, although larger than that found in adult tissues (75 versus 69 x 10(3) Mr), does not change during peri-implantation development. The size distribution of the intact proteoglycan does change, however, indicating developmental alterations in its glycosaminoglycan composition. These results indicate potential roles for syndecan in epithelial organization of the embryonic ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.     

Mesoderm induction in Xenopus laevis: responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells

When Xenopus embryos are cultured in calcium- and magnesium-free medium (CMFM), the blastomeres lose adhesion but continue dividing to form a loose heap of cells. If divalent cations are restored at the early gastrula stage the cells re-adhere and eventually form muscle (a mesodermal cell type) as well as epidermis. If, however, the cells are dispersed during culture in CMFM, muscle does not form following reaggregation although epidermis does. This suggests that culturing blastomeres in a heap allows the transmission of mesoderm-induction signals from cell to cell while dispersion effectively dilutes the signal. In this paper, we have attempted to substitute for cell proximity by culturing dispersed blastomeres in XTC mesoderm-inducing factor (MIF). We find that dispersed cells do not respond to XTC-MIF by forming mesodermal cell types after reaggregation, but the factor does inhibit epidermal differentiation. One interpretation of this observation is that an early stage in mesoderm induction is the suppression of epidermal differentiation and that formation of mesoderm may require contact-mediated signals that are produced in response to XTC-MIF. We have gone on to study the suppression of epidermal differentiation in more detail. We find that this is a dose-dependent phenomenon that can occur in single cells in the absence of cell division. Animal pole blastomeres become more difficult to divert from epidermal differentiation at later stages of development and by stage 12 they are 'determined' to this fate. Fibroblast growth factor (FGF) also suppresses epidermal differentiation in isolated animal pole blastomeres and transforming growth factor-beta 1 acts synergistically with FGF in doing so.     

Stage-related capacity for limb chondrogenesis in cell culture.

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm²). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.     

Changes in states of commitment of single animal pole blastomeres of Xenopus laevis

The experiments described in this paper were designed to compare the normal fates of animal pole blastomeres of Xenopus laevis with their state of commitment. Single animal pole blastomeres were labeled with a lineage marker and transplanted into the blastocoels of host embryos of different stages. The distribution of labeled daughter cells in the tadpole reflects the state of commitment of the parent cell at the time of transplantation. It is known that cells from the animal pole of the early blastula normally contribute predominantly to ectoderm with a small, but significant, contribution to the mesoderm. We show that on transplantation to the blastocoels of late blastula host embryos these blastomeres are pluripotent, contributing to all three germ layers. At later stages the normal fate of these cells becomes restricted solely to ectoderm and concomitantly the proportion of pluripotent cells is reduced, although the results depend upon the stage of the host embryo. Blastomeres from late blastula donors transplanted to mid gastrulae contribute solely to ectoderm in 34% of cases; however, in earlier hosts, when the vegetal hemisphere cells have "mesoderm inducing" or "vegetalizing" activity, late blastula animal pole blastomeres contribute to mesoderm and endoderm rather than ectoderm. Thus during the blastula stage animal pole cells pass from pluripotency to a labile state of commitment to ectoderm.     

Regionalization of the tail-tip epidermis requires inductive influence from vegetal cells and FGF signaling in the development of an ascidian, Halocynthia roretzi

The epidermis of an ascidian larva derived from animal-hemisphere cells is regionalized along the anterior-posterior (AP) axis through inductive signals emanating from vegetal-hemisphere cells in early stages of the development. Previously, we showed by blastomere isolation and ablation experiments that the contact between the animal and vegetal hemispheres until the 32-cell stage is necessary for the proper AP patterning of the epidermis in the tailbud-stage embryo. We here addressed the patterning mechanism of the posteriormost epidermis using a tail-tip epidermis marker, HrTT-1. Employing blastomere isolation and ablation experiments along with knockdown of a master regulator gene for posterior mesoderm, we have demonstrated that presence of the posterior vegetal cells after the 32-cell stage is necessary for the expression of HrTT-1. To explore the timing and nature of the influence of the posterior vegetal cells, we treated the embryos with FGF signaling inhibitors at various developmental stages and found that HrTT-1 expression was lost from embryos treated with the inhibitors from stages earlier than the late neurula stage, just prior to the onset of HrTT-1 expression but not after the initial tailbud stage, at which the expression of HrTT-1 had started. In embryos lacking HrTT-1 expression, the expression domain of Hrcad, which would otherwise be localized anterior to that of HrTT-1, expanded to the tail-tip. These results suggest that FGF signaling from the neurula to initial tailbud stages is necessary for the initiation but not maintenance of HrTT-1 expression in the tail-tip epidermis. The contact with posterior vegetal cells until and after the 32-cell stage may be required for FGF signaling to occur in the posterior tail, which in turn regionalizes the tail-tip epidermal territory.     

Basic fibroblast growth factor accumulates in the nuclei of various bFGF-producing cell types

The intracellular localization of basic fibroblast growth factor (bFGF) was studied in BHK-21 cells transfected with an expression vector containing the complementary DNA (cDNA) of the human bFGF gene (pbFGF). The intracellular location of bFGF was determined using indirect immunofluorescence. The antibodies used were polyclonal antibodies directed against either recombinant human bFGF or recombinant Xenopus bFGF. The nuclei of transfected cells that produce bFGF, but not the nuclei of untransfected cells, were labeled strongly by the antibodies. The nuclear staining was totally abolished when anti-bFGF antibodies preadsorbed with bFGF were used. Several types of endothelial cells known to produce bFGF were also stained in their nuclei by the antibodies. Nuclear extracts prepared from transfected cells were found to contain bFGF as determined using heparin-sepharose affinity chromatography, followed by Western blot analysis of fractions, which stimulated the proliferation BHK-21 cells. The mitogenic activity associated with the nuclei was not destroyed when isolated cell nuclei were digested by trypsin. It is therefore likely that the nucleus associated bFGF is intranuclear. These findings suggest that some biological activities of bFGF may be mediated by nuclear bFGF binding proteins or by the direct binding of bFGF to DNA.     

The marginal zone of the 32-cell amphibian embryo contains all the information required for chordamesoderm development.

The formation of the amphibian organizer is evidenced by the ability of cells of the dorsal marginal zone (DMZ) to self-differentiate to form notochord and to induce the formation of other axial structures from neighboring regions of the embryo. We have attempted to determine when these abilities are acquired in the urodele, Ambystoma mexicanum (axolotl), and in the anuran, Xenopus laevis, by removing the mesodermalizing influence of the vegetal hemisphere at different stages of development and culturing the animal hemisphere isolate. This was possible, even at the 32 and 64-cell stage, through the use of embryos with rare cleavage patterns. Cultured isolates were analyzed for morphological differentiation of mesodermal and neural structures, and for biochemical differentiation of the tissue-specific enzyme, acetylcholinesterase (AChE). Large amounts of mesodermal and neural structures, and normal expression of AChE were found in isolates made as early as the 32-cell stage in both species. Only a small increase in the percentage of isolates developing mesoderm was detected when isolations were made at later cleavage or blastula stages. The amount of mesoderm formed did not depend on the stage of isolation. Mesoderm differentiation was usually limited to the notocord and muscle. The isolates rarely formed pronephros, mesothelium, or mesenchyme, derivatives of ventral mesoderm, during normal development. The results indicate that the marginal zone of the cleavage-stage embryo contains all of the information needed for the formation of the organizer. The formation of dorsal mesoderm does not require subsequent interaction with the cells of the vegetal hemisphere, although the presence of those cells is likely to play a role in normal pattern formation.     

Clonal analysis of mesoderm induction in Xenopus laevis

Acidic fibroblast growth factor (aFGF) has been used to induce mesoderm from single animal pole cells of midblastula stage Xenopus embryos. The cells are individually cultured in a completely defined medium and are able to differentiate as small clones in a high proportion of cases. FGF-treated cells can give rise to several mesodermal cell types, while untreated cells show only epidermal or neural differentiation. Mesodermal differentiation can occur in clones of as few as eight cells, indicating that any additional cell-cell interactions required for mesodermal differentiation can be met by the medium used.     

小鼠早期胚胎发育期间TGF—β免疫组织化学定位

The distribution of transforming growth factor beta-1 (TGF-beta-1) in the early developing mouse embryos between day 1 and day 12 of gestation was examined by immunohistochemical techniques. Polyclonal rabbit antiserum raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta-1 from human platelets was used. The following results were obtained: 1. Embryonic cells of early cleavage stages (2, 4 and 8 cells) and late morulae showed positive immunofluorescent reaction without any difference in staining intensity (Plate I, Figs. 1-4). 2. Marked staining of blastocysts in toto or sections with anti-TGF-beta-1 antibodies by either immunofluorescence or immunoperoxidase reaction was also observed. Inner cell mass (ICM) cells and trophoectoderm cells were both reacted, but more intense staining was found in primary endoderm cells differentiated from ICM cells adjacent to blastocoele (Plate II, Fig. 5). 3. Scattered granules stained strongly with immunoperoxidase reaction were present in embryonic ectoderm and visceral endoderm surrounding the forming mesoderm which was only slightly stained (Plate II, Fig. 6). 4. Intense immunoperoxidase staining was also present in mesoderm of visceral yolk sac of day 8 and day 10 embryos (Plate II, Fig. 7). 5. During the formation of somites, neural tube and limb bud, remarkable staining was found in mesenchyme, individual cells of somites, mucous layer of gut tubes, heart and limb buds (Plate III, Figs. 8-10). No significant staining was seen in neural cells per se except the inner surface of neural tube. The results of present studies indicate that abundant TGF-beta-1 is present in preimplantation mouse embryos including cleavage, morulae and blastocyst stages. In postimplantation embryos, TGF-beta-1 appears to play an important role in the differentiation of endoderm and mesoderm, particularly in the development of extraembryonic tissues, and in later morphogenetic and histogenetic events involving mainly mesoderm or mesenchyme cells.     

The formation of primordial germ cells from germline cells in spherical embryos derived from the blastodisc of 2-cell embryos in goldfish, Carassius auratus

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nos1 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.     

Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.