Melanophore lineage and clonal organization of the epidermis in Xenopus embryos as revealed by expression of a biogenic marker, GFP

Melanophore lineage during embryogenesis of Xenopus laevis was traced using the overexpression of a biogenic marker, green fluorescent protein (GFP). Two different approaches were applied after injection of GFP mRNA (hence a marker construct) into each blastomere at the 16-cell stage. In in vivo experiments, the embryos injected with a marker construct were grown until stage 45, in which melanophores were distributed over the whole body and were good enough for checking GFP expression at their migratory destination. In in vitro experiments, neural tubes of the embryos injected with a marker construct were isolated and cultured at stage 21 to examine by virtue of GFP expression how neural crest cells differentiate into melanophores. The results obtained from both in vivo and in vitro experiments indicated the following: 1) selected animal blastomeres vastly contribute to the development of melanophores, whereas other animal blastomeres do so slightly at a limited pace; and 2) vegetal blastomeres never contribute to melanophores in normal development, whereas certain vegetal blastomeres have a potential to give rise to melanophores in vitro. The analyses using GFP also disclosed that the dorsal and ventral epidermis derive from the restricted animal blastomeres in the normal development. Since the dorso-ventrality of the epidermis has been inseparably coupled with integumental pigmentation, the clonal organization of the epidermis observed in the present study is discussed in the light of pigment pattern formation attributed by melanophores.     

Pattern regulation in defect embryos of Xenopus laevis

Defect embryos of 24 series were prepared by removing increasing numbers of blastomeres from an 8-cell embryo of Xenopus laevis. They were cultured and their development was examined macroscopically when controls reached a tailbud stage or later. Results show that most of defect embryos of 12 series develop normally, and some of them become normal frogs. Each of these defect embryos contain at least two animal blastomeres, one dorsal, and one ventral blastomere of the vegetal hemisphere. This suggests that a set of these four blastomeres of the three types is essential for complete pattern regulation.     

Use of Hybrids between Xenopus laevis and Xenopus borealis in Chimera Formation: Dorsalization of Ventral Cells

The combination of Xenopus borealis and X. laevis provides an excellent cell marking system. The potential availability of this system for chimera formation has also been suggested. However, eggs and early embryos of these species differ in size and the fusion of blastomeres of different sizez results in some disturbance in arrangement of blastomeres of a chimera. This disturbance was avoided by use of embryos from X. laevis eggs fertilized with X. borealis sperm, instead of X. borealis embryos. The cells of these hybrids could also be distinguished from the cells of X. laevis.
The fate of animal ventral cells placed in the dorsal region was followed by making a chimera by fusing a right lateral half of an 8-cell X. laevis embryo with that of an 8-cell hybrid embryo. The animal ventral cells in the "dorsal" region were found to become "dorsalized", giving rise to a lateral half of dorsal axial structures. This observation explains a previous finding that the replacement of dorsal cells by ventral ones had no effect on embryogenesis in a composite embryo.     

Pigment cell pattern formation in amphibian embryos: a reexamination of the dopa technique

Neural crest-derived melanophores form species-specific patterns in the dermis of amphibian embryos. Melanophore patterns may be generated by one of two general mechanisms: pigment cell precursors disperse throughout the embryo, with melanophores differentiating in certain regions due to environmental cues, or melanoblasts may localize in different regions as a result of a hierarchy of tissue affinities. Both of these mechanisms have been proposed to be responsible for the dorso-ventral patterning of melanophores in Xenopus laevis. We have reexamined the distribution of melanoblasts in X. laevis and Taricha torosa using the dopa (3,4-dihydroxyphenyl-alanine)-staining technique. We have found that many of the dopa-positive cells identified as melanoblasts by some researchers are actually not derived from the neural crest: dopa-positive cells in T. torosa were identified in the transmission electron microscope to be either leukocytes or erythrocytes, in X. laevis dopa-positive cells are found between the ectoderm and somites where neural crest cells are not found, and X. laevis embryos surgically depleted of neural crest have dopa-staining patterns identical to control embryos. Melanoblasts are apparently not found in the ventralmost regions of early T. torosa and X. laevis embryos, providing additional evidence for the role of differential tissue affinities in directing the formation of embryonic pigment cell patterns.     

Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.     

Mechanisms of Melanophore Induction in Amphibian Development

Induction of melanophores was examined by the sandwich method of explantation with embryonic tissues of Xenopus laevis +/+ and the white mutant, a^P/a^P. Interspecific combinations of tissues of Triturus taeniatus and Xenopus borealis were also used. The ectoderm used as the reacting system was taken from embriyos at various stages and combined with various tissues known to be melanogenic inductors. The following results were obtained: 1) The sources of melanophore induction in both +/+ and a^p/a^p studied by sandwich explantation were the same in both retinal pigmented epithelium and dermal melanophores: 2) Melanophores were induced in epidermal material from embryos at stages from the early gastrula to the late tail bud stage: 3) The presence of melanoblasts together with other ectomesenchymal cells in the neural crest is not sine qua non for their determination and differentiation: 4) On isolation of reacting material from the late gastrula, melanophores appeared in all cases. This shows that two hours contact between inductor tissues and the ectoderm is necessary and sufficient for melanophore induction: 5) Melanophore induction is not species-specific, but occurred in Xenopus ectoderm under the action of endomesoderm of Tr. taeniatus or X. borealis , and vice versa. The shapes and structures of melanophores induced were typical for the species from which the ectoderm was taken: 6) Melanogenic activity in the late gastrula stage has a gradient of distribution with a maximum in the prechordal plate: 7) In the mutant only the primary source of melanogenic inductors, the prechordal plate (PrP1), was active in stages both before and after its invagination: 8) Despite the fact that skin melanophores and retinal melanocytes have different genesis in development, all the present data suggest the identity of the mechanisms of melanin synthesizing machinery in the two.     

Fates of the blastomeres of the 32-cell-stage Xenopus embryo

A detailed fate map of all of the progeny derived from each of the blastomeres of the 32-cell-stage South African clawed frog embryo (Xenopus laevis), which were selected for stereotypic cleavages, is presented. Individual blastomeres were injected with horseradish peroxidase and all of their descendants in the late tailbud embryo (stages 32 to 34) were identified after histochemical processing of serial tissue sections and whole-mount preparations. The progeny of each blastomere were distributed characteristically, both in phenotype and location. Most organs were populated largely by the descendants of particular sets of blastomeres, the progeny of each often being restricted to defined spatial addresses. Thus, the descendants of any one blastomere were distinct and predictable when embryos were preselected for stereotypic cleavages. However, variations among embryos were common and the frequencies with which one may expect organs to contain progeny from any particular blastomere are reported. The differences in the fates of the 16-cell-stage blastomeres and their 32-cell-stage daughter blastomeres are outlined and can be grouped into three general categories. The two daughter cells may give rise to equal numbers of cells in a particular organ, one daughter cell may give rise to many more of the cells in an organ derived from the mother blastomere, or one daughter cell may give rise to all of the progeny in an organ derived from the mother blastomere. Thus, cell fates are segregated during cleavage stages in both symmetric and asymmetric manners, and the lineages exhibit a diversification mode (G. S. Stent, 1985, Philos. Trans R. Soc. London Ser. B 312, 3-19) of cell division.     

Differential participation of ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus, as revealed in diploid-triploid or interspecific chimeras

The thymocytes in the early larvae of Xenopus laevis have been shown to be derived from precursor cells immigrating interstitially through the mesenchyme into the organ rudiments at 3-4 days of age (Nieuwkoop and Faber stages 42-45). Orthotopic grafting of diploid tissues onto triploid stage 22 embryos followed by ploidy analyses of their hemopoietic cells revealed that both thymocytes and erythrocytes in early larvae are derived from the ventral blood islands (VBI), whereas those in late larvae and adults come mainly from the dorsolateral plate (DLP). To study how the VBI cells of embryos at stage 22 participate in hemopoiesis, a number of interspecific chimeras were produced in X. laevis and X. borealis embryos. Sections of the chimeras at various developmental stages were examined by employing the unique stainability of X. borealis nuclei to quinacrine as a marker; the results show that the VBI-derived cells enter into the circulation around stage 35/36, and that some of them leave the blood vessels to migrate interstitially through the mesenchyme toward the thymic rudiment during stages 43-45. A minor population of the VBI-derived cells was also found extravascularly in the mesonephric primordia. In contrast to the VBI, the DLP-derived cells contributed to the hemopoietic cell population not in early larvae, but in late ones as a major constituent in the mesonephros, thymus, liver, and peripheral blood.     

Studies on cellular adhesion of Xenopus laevis melanophores: pigment pattern formation and alteration in vivo by endogenous galactoside-binding lectin or its sugar hapten inhibitor

We have studied the development of Xenopus laevis tail melanophores and the effects on these cells on confrontation with endogenous X. laevis galactoside-binding lectin or its sugar hapten inhibitor thiodigalactoside (TDG). An initial population of unpigmented cells differentiates into melanophores on the dorsal surface of the neural tube, and on the dorsal and ventral apices of the myotomes, forming the larval pattern. Melanophores secondarily populate the flank, forming a spaced arrangement which is later transformed into a dorsal and ventral strip. A technique has been developed for confrontation of premigratory neural crest with purified lectin or TDG. These molecules impact on tail melanophores. With lectin treatment melanophore numbers decrease, and cell morphologies and arrangements change. TDG treatment, however, primarily affects pigment cell morphology. These results suggest that both galactoside-bearing receptors for this lectin and the lectin itself can affect melanophores in this species of frog.     

Early patterning of the mouse embryo--contributions of sperm and egg

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.     

Contribution of Cadherins to Directional Cell Migration and Histogenesis in Xenopus Embryos

Perturbation of adhesion mediated by cadherins was achieved by over-expressing truncated forms of E- and EP-cadherins (in which the extracellular domain was deleted) in different blastomeres of stage 6 Xenopus laevis embryos. Injections of mRNA encoding truncated E- and EP-cadherins into A1A2 blastomeres resulted in inhibition of cell adhesion and, at later stages, in morphogenetic defects in the anterior neural tissues to which they mainly contribute. In addition, truncated EP-cadherin mRNA produced a duplication of the dorso-posterior axis in a significant number of cases. The expression of truncated E- and EP-cadherins in blastomeres involved in gastrulation and neural induction (B1B2 and C1), led to the duplication of the dorso-posterior axis as well as to defects in anterior structures. Morphogenetic defects obtained with truncated EP-cadherin were more severe than those induced with truncated E-cadherin. Cells derived from blastomeres injected with truncated EP-cadherin mRNA, dispersed more readily at the blastula and gastrula stages than the cells derived from the blastomeres expressing truncated E-cadherin. Presumptive mesodermal cells expressing truncated cadherins did not engage in coherent directional migration. The alteration of cadherin-mediated cell adhesion led directly to the perturbation of the convergent-extension movements during gastrulation as shown in the animal cap assays and indirectly to perturbation of neural induction. Although the cytoplasmic domains of type I cadherins share a high degree of sequence identity, the over-expression of their cytoplasmic domains induces a distinct pattern of perturbations, strongly suggesting that in vivo, each cadherin may transduce a specific adhesive signal. These graded perturbations may in part result from the relative ability of each cadherin cytoplasmic domain to titer the P-catenin.     

Contribution of Cadherins to Directional Cell Migration and Histogenesis in Xenopus Embryos

Perturbation of adhesion mediated by cadherins was achieved by over-expressing truncated forms of E- and EP-cadherins (in which the extracellular domain was deleted) in different blastomeres of stage 6 Xenopus laevis embryos. Injections of mRNA encoding truncated E- and EP-cadherins into A1A2 blastomeres resulted in inhibition of cell adhesion and, at later stages, in morphogenetic defects in the anterior neural tissues to which they mainly contribute. In addition, truncated EP-cadherin mRNA produced a duplication of the dorso-posterior axis in a significant number of cases. The expression of truncated E- and EP-cadherins in blastomeres involved in gastrulation and neural induction (B1B2 and C1), led to the duplication of the dorso-posterior axis as well as to defects in anterior structures. Morphogenetic defects obtained with truncated EP-cadherin were more severe than those induced with truncated E-cadherin. Cells derived from blastomeres injected with truncated EP-cadherin mRNA, dispersed more readily at the blastula and gastrula stages than the cells derived from the blastomeres expressing truncated E-cadherin. Presumptive mesodermal cells expressing truncated cadherins did not engage in coherent directional migration. The alteration of cadherin-mediated cell adhesion led directly to the perturbation of the convergent-extension movements during gastrulation as shown in the animal cap assays and indirectly to perturbation of neural induction. Although the cytoplasmic domains of type I cadherins share a high degree of sequence identity, the over-expression of their cytoplasmic domains induces a distinct pattern of perturbations, strongly suggesting that in vivo, each cadherin may transduce a specific adhesive signal. These graded perturbations may in part result from the relative ability of each cadherin cytoplasmic domain to titer the P-catenin.     

Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.     

Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90° before first cleavage

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90° with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35–38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90° rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90°, but the fate of the blastomeres did not simply show a 90° switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position; (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy

The Xenopus borealis quinacrine marker and scanning electron microscopy have been used to study the appearance, migration, and homing of neural crest cells in the embryo of Xenopus. The analysis shows that the primordium of the neural crest develops from the nervous layer of the ectoderm and consists of three segments at early neurula stages. This primordium is located in the lateral halves of the neural folds behind the prospective eye vesicles. The histological and experimental evidence shows that the neural crest cells also originate from the medial portion of the neural folds. The neural crest segments in the cephalic region start to migrate just before the closure of the neural tube. Isotopic and isochronic unilateral grafts of X. borealis neural crest into X. laevis embryos were performed in order to map the fate of the cranial crest segments and the vagal-truncal neural crest. The analysis of the X. laevis host embryos shows that the mandibular crest segment contributes to the lower jaw (Meckel's cartilage), quadrate, and ethmoid-trabecular cartilages, as well as to the ganglionic and Schwann cells of the trigeminus nerve, the connective tissues, the mesenchymal and choroid layers of the eye, and the cornea. The hyoid crest segment is located in the ceratohyal cartilage and in ganglia VII and VIII. The branchial crest segment migrates from the caudal part of the otic vesicle and divides into two portions which contribute to the cartilages of the gills. The vagal-truncal neural crest starts to migrate later at stage 25. It migrates by means of the vagus complex in a ventral direction and penetrates into the splanchnic layer of the digestive tract. The trunk neural crest cells disperse into three different pathways which differ from those of the avian embryo at this level.     

Maxadilan activates PAC1 receptors expressed in Xenopus laevis xelanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long-term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non-transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

Nuclear transplantation of mouse fetal germ cells into enucleated two-cell embryos

Mouse fetal germ cells were fused with enucleated blastomeres of two-cell embryos. Donor germ cells were obtained from fetuses of albino CD-1 strain or pigmented F(1) (C57BL x CBA) female mice mated with the same strain males at 11.5 to 16.5 days post coitum. Recipient two-cell embryos, which were of a different strain from the donors, were obtained at 37 to 42 hours (Group 1), 42 to 47 hours (Group 2), and 47 to 52 hours (Group 3) after treatment with human chorionic gonadotropin (hCG). After removing the nucleus from one two-cell blastomere, a single germ cell was fused with the enucleated blastomere using the Sendai virus; the second blastomere was left intact. The reconstituted embryos were cultured for 3 days in vitro, to examine their developmental capacity. The fused blastomeres in Groups 1 and 2 did not divide, but a few transplanted blastomeres in Group 3 divided several times, and some of them developed into normal blastocysts. Most embryos developed into blastocysts from one blastomere, with an undivided blastomere remaining. Embryos developing into normal blastocysts or blastocysts with small blastomeres were transferred to the oviducts of Day-1 or the uteri of Day-3 pregnant albino CD-1 mice. None of the young showed any contribution of the germ cells, judging by the eye and coat colors and by the germ cells in the germ line following mating with albino mice. Possible reasons for failure of pluripotency of the germ cells are discussed here.