Isolation of a Factor Mediating Melanogenic Induction in Pigment Cells of Xenopus Laevis

An activity has been identified in +/+ Xenopus laevis embryonic extract which stimulates intensive and stable melanoprotein synthesis in retinal pigmented epithelium and dermal melanophores of albino periodical mutants, a^p/a^p. Experiments involved four steps: 1) preparation of extract from +/+ embryos at stages NF 18–21 when melanogenesis occurs most extensively; 2) gel-filtration of the extract; 3) microinjection of the fractionated materials into a^p/a^p; and 4) identification of the nature of active substances. The activity is heat-labile and is destroyed by treatment with pronase E. We call this material melanogenic factor (MGF) and suggest that it is responsible for initiating melanogenic activity in melanin-synthesizing cells.     

Homoiogenetic Neural Induction in Xenopus Chimeric Explants

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis , either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.     

Distribution of differentiation potentials and the conditions for their realization in the amphibian neuroectoderm

The X. laevis neuroectoderm (NE) at the mid and late gastrula stages is capable to form mesoderm in vitro after its separation from mesoderm. This capacity is inherent in posterior 2/3 of NE underlied by axial mesoderm in the embryo and forming deuterencephalic and trunk regions of the brain in the normal development. The archencephalic 1/3 of NE of the late gastrula, underlied in the embryo by prechordal plate, is capable of differentiation into archencephalic regions of the brain, rather than into mesoderm. For the typical differentiation of archencephalic NE to be realized, it should be surrounded by the outer ectoderm layer. In the absence of the latter, the whole explant develops into retina and brain only. Inside the closed explants, ectomesenchyme and melanophores arise and the eye material is subdivided into retina and pigmented epithelium. The archencephalic NE, dissociated to individual cells and wrapped into epidermis, forms much more ectomesenchyme and melanophores than the usual NE explants.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

Vertical versus planar induction in amphibian early development

In the Urodeles, the archenteron roof invaginates as a single continuous sheet of cells, vertically inducing the neural anlage in the overlying ectoderm during invagination. The induction comprises first the activation process, leading, to forebrain differentiation tendencies, and then the superimposed transformation process, which changes presumptive forebrain development into that of hindbrain and spinal cord acting with a caudally increasing intensity. The activating action, being maximal anteriorly, decreases caudally to nearly zero. In the double-layered Xenopus embryo, the internal mesodermal marginal zone shows much more independent and earlier regional segregation and involution than the external marginal zone in the Urodeles; its prechordal mesoderm already initiating vertical neural induction in overlying ectoderm at stages 10 to 10⁺ before any visible archenteron invagination. In Xenopus incomplete exogastrulae the prechordal mesoderm involutes normally prior to evagination of the endoderm and mesodem. Artificially produced Xenopus total exogastrulae, made at stage 9 before mesoderm involution, behave just like axolotl total exogastrulae, showing no neural differentiation. The notion of planar neural induction in Xenopus can only be applied in exogastrulae and Keller explants for the transforming action, which is maximal in the caudal archenteron roof. In normal Xenopus development, the formation of the entire nervous system is essentially due to vertical induction by the successively involuting prechordal and notochordal mesoderm. The different behavior of Xenopus embryos in comparison with Urodele embryos can essentially be explained by the double-layered character of the animal moiety of the Xenopus embryo.     

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus

We examined the timing and mechanisms of mesodermal and neural determination in Cynops , using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops .     

Anterior endoderm and head induction in early vertebrate embryos

Early work on the formation of the vertebrate body axis indicated the existence of separate head- and trunk-inducing regions in Spemann's organizer of the amphibian gastrula. In mammals some head-organizing activity may be located in anterior visceral (extraembryonic) endoderm (AVE). By analogy, the equivalent structure in the Xenopus laevis gastrula, the anterior endoderm, has been proposed to be the amphibian head organizer. Here we review recent data that challenge this notion and indicate that the involvement of AVE in head induction seems to be an exclusively mammalian characteristic. In X. laevis and chick, it is the prechordal endomesoderm that is the dominant source of head-inducing signals during early gastrulation. Furthermore, head induction in mammals needs a combination of signals from anterior primitive endoderm, prechordal plate, and anterior ectoderm. Thus, despite the homology of vertebrate anterior primitive endoderm, a role in head induction seems not to be conserved.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Embryonic induction and cation concentrations in amphibian embryos

Explanted ectoderm from early gastrulae of Triturus alpestris was treated with the Na-K ionophore gramicidin (10(-9) to 10(-5) M) and the Ca-ionophore A 23187 (10(-7) to 10(-5) M). The ectoderm developed almost exclusively to atypical epidermis as in the control explants. When the ectoderm was treated with ouabain (10(-4) M), intracellular Na+ increased about 4.4-fold and K+ was reduced by half. Mesenchyme cells in small number differentiated in about 40% of the ouabain-treated explants. The time course of total Na+ and K+ ion concentrations was measured over a period of 72 h in ectoderm of T. alpestris after induction with vegetalizing factor and in control explants. In the first 15 h after explantation, no significant differences between control and induced explants were found. Thereafter, the steady state concentration of K+ decreased in the induced explants, whereas the steady-state concentration of Na+ slightly increased. The membrane resting potential recorded intracellularly of ectoderm sandwiches from early gastrula stages was found to be -41.3 mV in control and -59.3 mV in induced explants. From the specific conductances and permeabilities of non-induced and induced cells it is concluded that the induction process leads to a differentiation of the cell membrane, which acquires the characteristics of ionic selectivity. Ectoderm from Ambystoma mexicanum forms neural or neuroid tissue, mesenchyme and melanophores after explantation in salt solution in up to 50% of the explants without any additions. Isolated Ambystoma ectoderm is therefore not suitable for test experiments.     

Embryonic Appearance of α, β, and γ Crystallins in the Periodic Albinism (ap) Mutant of Xenopus laevis

The appearance of the crystallins during lens development in the periodic albinism (a^p/a^p) mutant of Xenopus laevis has been studied. Using antibodies specific for total crystallins, α+β crystallins, and γ crystallins in the immunofluorescence technique, the first positive reaction for all could be demonstrated in the Nieuwkoop-Faber Stage 31 lens rudiment. The antibody to α+β crystallins exhibited differences in intensity from cell to cell in the early rudiment, while the reaction to the other antibodies was uniform throughout the rudiment. As lens differentiation progressed, immunofluorescence was restricted in all cases to the lens fiber area, up to and including Nieuwkoop-Faber Stage 45. The lens epithelium of the one-year-old adult a^p/a^p was positive, however, for total lens crystallins.
These results are at variance with earlier studies on lens development and the crystallins in wild-type (+/+) X. laevis , where a positive reaction for y and total crystallins could be detected earlier, and in the lens epithelium of Nieuwkoop-Faber Stage 41 embryos for total lens crystallins. That this divergence in the mutant is due to a pleiotropic effect or directly to the inductive failure of the endomesoderm to initiate melanogenesis, is discussed.     

A homeobox-containing marker of posterior neural differentiation shows the importance of predetermination in neural induction

A homeobox sequence has been used to isolate a new Xenopus cDNA, named XIHbox6. A short probe from this gene serves as an early marker of posterior neural differentiation in the Xenopus nervous system. The gene recognized by this cDNA sequence is first transcribed at the late gastrula stage and solely in the posterior neural cells. The gene is expressed when ectodermal and mesodermal tissues of an early gastrula are placed in contact, but not by either tissue cultured on its own. However, gene expression is most easily inducible in ectoderm from the dorsal region, i.e., in ectoderm normally destined to form neural structures. This establishes the principle, in contrast to previous belief, that the induction of the embryonic nervous system involves a predisposition of the ectoderm and does not depend entirely on an interaction with inducing mesoderm.     

Inductive differentiation of two neural lineages reconstituted in a microculture system from Xenopus early gastrula cells.

Neural induction of ectoderm cells has been reconstituted and examined in a microculture system derived from dissociated early gastrula cells of Xenopus laevis. We have used monoclonal antibodies as specific markers to monitor cellular differentiation from three distinct ectoderm lineages in culture (N1 for CNS neurons from neural tube, Me1 for melanophores from neural crest and E3 for skin epidermal cells from epidermal lineages). CNS neurons and melanophores differentiate when deep layer cells of the ventral ectoderm (VE, prospective epidermis region; 150 cells/culture) and an appropriate region of the marginal zone (MZ, prospective mesoderm region; 5-150 cells/culture) are co-cultured, but not in cultures of either cell type on their own; VE cells cultured alone yield epidermal cells as we have previously reported. The extent of inductive neural differentiation in the co-culture system strongly depends on the origin and number of MZ cells initially added to culture wells. The potency to induce CNS neurons is highest for dorsal MZ cells and sharply decreases as more ventrally located cells are used. The same dorsoventral distribution of potency is seen in the ability of MZ cells to inhibit epidermal differentiation. In contrast, the ability of MZ cells to induce melanophores shows the reverse polarity, ventral to dorsal. These data indicate that separate developmental mechanisms are used for the induction of neural tube and neural crest lineages. Co-differentiation of CNS neurons or melanophores with epidermal cells can be obtained in a single well of co-cultures of VE cells (150) and a wide range of numbers of MZ cells (5 to 100). Further, reproducible differentiation of both neural lineages requires intimate association between cells from the two gastrula regions; virtually no differentiation is obtained when cells from the VE and MZ are separated in a culture well. These results indicate that the inducing signals from MZ cells for both neural tube and neural crest lineages affect only nearby ectoderm cells.     

Origin of the prechordal plate and patterning of the anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster

We analyzed the notochord formation, formation of the prechordal plate, and patterning of anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster. The lower (LDMZ) and upper (UDMZ) domains of the dorsal marginal zone (DMZ) of the early gastrula involuted and formed two distinct domains: the anterior fore-notochordal endodermal roof and the posterior domain containing the prospective notochord. Cygsc is expressed in the LDMZ from the onset of gastrulation, and the Cygsc-expressing LDMZ planarly induces the notochord in the UDMZ at the early to mid gastrula stages. At the mid to late gastrula stages, part of the Cygsc-expressing LDMZ is confined to the prechordal plate. On the other hand, Cybra expression only begins at mid gastrula stage, coincident with notochord induction at this stage. Anteroposterior regional specificity of the neural plate was patterned by the posterior domain of the involuting archenteron roof containing the prospective notochord at the mid to late gastrula stages. Cynops gastrulation thus differs significantly from Xenopus gastrulation in that the regions of the DMZ are specified from the onset of gastrulation, while the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Thus we propose that Cynops gastrulation is divided into two phases: a notochord induction phase in the early to mid gastrula, and a neural induction phase in the mid to late gastrula.     

Inductive effect of the eye tissues of adult clawed toads on the gastrula ectoderm

The inducing influence of adult eye tissues on the early gastrula ectoderm was studied in vitro. Both retina and pigment epithelium induced in the early gastrula ectoderm similar spectra of cell types, including nervous tissue, retina, pigment epithelium, lentoids, ectomesenchyme, and melanophores. It is suggested that the correspondence of these cell types with those arising at a spontaneous transdifferentiation of the isolated retina and pigment epithelium cells in vitro or at the induction of the early gastrula ectoderma by archencephalic endomesoderm during the normal development can be accounted for by that in these eye cells molecular determinants appeared as a result of induction and maintaina the stability of their differentiation and their potencies to transdifferentiation in vitro being reproduced during the lifetime of these cells.     

FGF signaling and the anterior neural induction in Xenopus

We previously showed that FGF was capable of inducing Xenopus gastrula ectoderm cells in culture to express position-specific neural markers along the anteroposterior axis in a dose-dependent manner. However, conflicting results have been obtained concerning involvement of FGF signaling in the anterior neural induction in vivo using the same dominant-negative construct of Xenopus FGF receptor type-1 (delta XFGFR-1 or XFD). We explored this issue by employing a similar construct of receptor type-4a (XFGFR-4a) in addition, since expression of XFGFR-4a was seen to peak between gastrula and neurula stages, when the neural induction and patterning take place, whereas expression of XFGFR-1 had not a distinct peak during that period. Further, these two FGFRs are most distantly related in amino acid sequence in the Xenopus FGFR family. When we injected mRNA of a dominant-negative version of XFGFR-4a (delta XFGFR-4a) into eight animal pole blastomeres at 32-cell stage, anterior defects including loss of normal structure in telencephalon and eye regions became prominent as examined morphologically or by in situ hybridization. Overexpression of delta XFGFR-1 appeared far less effective than that of delta XFGFR-4a. Requirement of FGF signaling in ectoderm for anterior neural development was further confirmed in culture: when ectoderm cells that were overexpressing delta XFGFR-4a were cocultured with intact organizer cells from either early or late gastrula embryos, expression of anterior and posterior neural markers was inhibited, respectively. We also showed that autonomous neuralization of the anterior-type observed in ectoderm cells that were subjected to prolonged dissociation was strongly suppressed by delta XFGFR-4a, but not as much by delta XFGFR-1. It is thus indicated that FGF signaling in ectoderm, mainly through XFGFR-4, is required for the anterior neural induction by organizer. We may reconcile our data to the current "neural default model," which features the central roles of BMP4 signaling in ectoderm and BMP4 antagonists from organizer, simply postulating that the neural default pathway in ectoderm includes constitutive FGF signaling step.     

Early neural crest induction requires an initial inhibition of Wnt signals

Neural crest (NC) induction is a long process that continues through gastrula and neurula stages. In order to reveal additional stages of NC induction we performed a series of explants where different known inducing tissues were taken along with the prospective NC. Interestingly the dorso-lateral marginal zone (DLMZ) is only able to promote the expression of a subset of neural plate border (NPB) makers without the presence of specific NC markers. We then analysed the temporal requirement for BMP and Wnt signals for the NPB genes Hairy2a and Dlx5, compared to the expression of neural plate (NP) and NC genes. Although the NP is sensitive to BMP levels at early gastrula stages, Hairy2a/Dlx5 expression is unaffected. Later, the NP becomes insensitive to BMP levels at late gastrulation when NC markers require an inhibition. The NP requires an inhibition of Wnt signals prior to gastrulation, but becomes insensitive during early gastrula stages when Hairy2a/Dlx5 requires an inhibition of Wnt signalling. An increase in Wnt signalling is then important for the switch from NPB to NC at late gastrula stages. In addition to revealing an additional distinct signalling event in NC induction, this work emphasizes the importance of integrating both timing and levels of signalling activity during the patterning of complex tissues such as the vertebrate ectoderm.     

Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation

Vertebrate neural induction requires inhibition of bone morphogenetic protein (BMP) signaling in the ectoderm. However, whether inhibition of BMP signaling is sufficient to induce neural tissues in vivo remains controversial. Here we have addressed why inhibition of BMP/Smad1 signaling does not induce neural markers efficiently in Xenopus ventral ectoderm, and show that suppression of both Smad1 and Smad2 signals is sufficient to induce neural markers. Manipulations that inhibit both Smad1 and Smad2 pathways, including a truncated type IIB activin receptor, Smad7 and Ski, induce early neural markers and inhibit epidermal genes in ventral ectoderm; and co-expression of BMP inhibitors with a truncated activin/nodal-specific type IB activin receptor leads to efficient neural induction. Conversely, stimulation of Smad2 signaling in the neural plate at gastrula stages results in inhibition of neural markers, disruption of the neural tube and reduction of head structures, with conversion of neural to neural crest and mesodermal fates. The ability of activated Smad2 to block neural induction declines by the end of gastrulation. Our results indicate that prospective neural cells are poised to respond to Smad2 and Smad1 signals to adopt mesodermal and non-neural ectodermal fates even at gastrula stages, after the conventionally assigned end of mesodermal competence, so that continued suppression of both mesoderm- and epidermis-inducing Smad signals leads to efficient neural induction.     

Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.     

Neural Induction by Treatment with Ca2+-free Saline Solution in the Gastrula Ectoderm of Cynops pyrrhogaster and Inhibition of Neural Induction with Fetal Calf Serum

The relation between the inducing activity and the cell-dissociation effect of Ca²⁺-free (or Ca²⁺, Mg²⁺-free) saline solution (CF or CMF) on the early gastrula ectoderm was examined. In the culture medium containing no fetal calf serum (FCS), most ectoderm cells treated with CF or CMF died within a few days and only a few differentiated into epidermal cells. However, when the culture medium contained 2% FCS, ectoderm cells treated with CF or CMF differentiated into neural crest derivatives (NCDs), such as mesenchyme cells, pigment cells, and nerve cells. The frequency of the induction depended only on the duration of CF- or CMF-treatment. FCS alone had no inducing activity on ectoderm cells. On the contrary a high concentration of FCS gave an inhibitory effect on the induction. These results indicate that CF is a neuralizing factor and that CF-treated cells require FCS, not for induction, but for survival and differentiation. With CF, the maximum induction of NCDs required a longer duration than that necessary for complete cell-dissociation. This result suggests that the induction depends on some effects of CF other than cell-dissociation.     

Expression of N-CAM precedes neural induction in Pleurodeles waltl (urodele, amphibian)

The appearance and localization of N-CAM during neural induction were studied in Pleurodeles waltl embryos and compared with recent contradictory results reported in Xenopus laevis. A monoclonal antibody raised against mouse N-CAM was used. In the nervous system of Pleurodeles, it recognized two glycoproteins of 180 and 140x10(3) M(r) which are the Pleurodeles equivalent of N-CAM-180 and -140. Using this probe for immunohistochemistry and immunocytochemistry, we showed that N-CAM was already expressed in presumptive ectoderm at the early gastrula stage. In late gastrula embryos, a slight increase in staining was observed in the neurectoderm, whereas the labelling persisted in the noninduced ectoderm. When induced ectodermal cells were isolated at the late gastrula stage and cultured in vitro up to 14 days, a faint polarized labelling of cells was observed initially. During differentiation, the staining increased and became progressively restricted to differentiating neurons.