INDUCTION OF THE FROG HATCHING GLAND CELL FROM EXPLANTED PRESUMPTIVE ECTODERMAL TISSUE BY LICL

When cells of the superficial layer explanted from the presumptive ectoderm of a Rana japonica early gastrula embryo at stage 10 were cultured in standard salt solution for 4–7 days, they differentiated into cement gland cells (CGCs), cilia cells (CCs) and common epidermal cells (CECs). When, however, these explants were treated with LiCl and transferred to Barth's solution, hatching gland cells (HGCs) and pigment cells were induced.
The optimum condition for inducing differentiation of HGC was treatment with 70 mM LiCl for 6–8 hr at 18°C. The best ability to react to the HGC-inducing stimuli resided in the superficial layer of the dorsal presumptive epidermis of the embryo at stage 10. Upon repeated stimulation, explants from stage 8 embryos underwent differentiation into nerve and pigment cells, whereas those from stage 11 embryos differentiated into CCs and CECs. Under optimum conditions, the total volume of HGCs induced amounted to about 70% of the explanted tissue. The culture media from LiCl-induced HGCs showed an apparent jelly-digesting activity, strongly indicating that the cells were functionally identical with those differentiated in situ .     

Expression of estrogen induced gene 121-like (EIG121L) during early Xenopus development

Estrogen induced gene 121 (EIG121) and EIG121-like (EIG121L) are evolutionarily conserved genes. But, their function is still unknown. Here, we report the expression pattern of Xenopus EIG121-like (xEIG121L) during early development. Its expression was first detected at stage 9 after mid-blastula transition, attained its maximal level at the gastrula stage, and remained constant until the tadpole stage. Whole-mount in situ hybridization revealed that xEIG121L was expressed strongly in the ventral ectoderm at the gastrula stage, and in the anterior ectoderm surrounding the neural plate at the neurula stage. xEIG121L expression was especially high in the presumptive hatching gland and cement gland regions in the neurula. At the tailbud stage, xEIG121L expression was limited to the hatching gland; an inverted Y type staining, characteristic of the hatching gland, was observed. However, at the tadpole stage, xEIG121L was expressed broadly in the head, heart and fin.     

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.     

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.     

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.     

Mesoderm induction in the future tail region of Xenopus

Summary We have used interspecific grafts between Xenopus borealis and Xenopus laevis to study the signalling system that produces tail mesoderm. Early gastrula ectoderm grafted into the posterior neural plate region of neurulae responds to a mesodermal inducing signal in this region and forms mainly tail somites; this signal persists until at least the early tail bud stage. Ventral ectoderm grafted into the posterior neural plate loses its competence to respond to this signal after stage 10 1/2. We have established the specification of anterior and posterior neural plate ectoderm. In ectodermal sandwiches or when grafted into unusual positions, anterior regions gave rise to mainly nervous system and posterior regions to large amounts of muscle, together with some nervous system. Thus it was impossible to assess the competence of posterior neural plate ectoderm to form further mesoderm and hence to establish if mesodermal induction continues during neurulation in unmanipulated embryos.     

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.     

SCANNING ELECTRON MICROSCOPICAL APPEARANCE OF CELLS FROM XENOPUS LAEVIS EMBRYOS OF DIFFERENT STAGES

The scanning electron microscopical appearances of cells isolated from different regions of Xenopus laevis embryos of different stages, and cultured in vitro have been compared. Blastula inner ectoderm cells initially show filopodia, then become flattened onto the substrate and then form pseudopodia. Blastula outer ectoderm cells are initially similar, but do not form pseudopodia. Most of the ectoderm cells from gastrulae and neurulae are featureless. Endoderm cells from blastulae do not initially form filopodia, but later form pseudopodia. Most of the endoderm cells from gastrulae and neurulae show neither filopodia nor pseudopodia, but in the gastrula some elongated, cylindrical cells are observed. Thus cells change their appearance after the three hour culture period; cells from different regions of embryos of the same stage show different appearances in vitro ; and cells from equivalent regions of embryos of different stages show different behaviours in vitro.     

<Emphasis Type="Italic">AmphiFoxQ2</Emphasis>, a novel winged helix/forkhead gene,exclusively marks the anterior end of the amphioxus embryo

A full-length FoxQ-related gene (AmphiFoxQ2) was isolated from amphioxus. Expression is first detectable in the animal/anterior hemisphere at the mid blastula stage. The midpoint of this expression domain coincides with the anterior pole of the embryo and is offset dorsally by about 20 degrees from the animal pole. During the gastrula stage, expression is limited to the anterior ectoderm. By the early neurula stage, expression remains in the anterior ectoderm and also appears in the adjacent anterior mesendoderm. By the early larval stages, expression is detectable in the anteriormost ectoderm and in the rostral tip of the notochord. AmphiFoxQ2 is never expressed anywhere except at the anterior tip of amphioxus embryos and larvae. This is the first gene known that exclusively marks the anterior pole of chordate embryos. It may, therefore, play an important role in establishing and/or maintaining the anterior/posterior axis.     

Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.

The ability of a tissue to respond to induction, termed its competence, is often critical in determining both the timing of inductive interactions and the extent of induced tissue. We have examined the lens-forming competence of Xenopus embryonic ectoderm by transplanting it into the presumptive lens region of open neural plate stage embryos. We find that early gastrula ectoderm has little lens-forming competence, but instead forms neural tissue, despite its location outside the neural plate; we believe that the transplants are being neuralized by a signal originating in the host neural plate. This neural competence is not localized to a particular region within the ectoderm since both dorsal and ventral portions of early gastrula ectoderm show the same response. As ectoderm is taken from gastrulae of increasing age, its neural competence is gradually lost, while lens competence appears and then rapidly disappears during later gastrula stages. To determine whether these developmental changes in competence result from tissue interactions during gastrulation, or are due to autonomous changes within the ectoderm itself, ectoderm was removed from early gastrulae and cultured for various periods of time before transplantation. The loss of neural competence, and the gain and loss of lens competence, all occur in ectoderm cultured in vitro with approximately the same time course as seen in ectoderm in vitro. Thus, at least from the beginning of gastrulation onwards, changes in competence occur autonomously within ectoderm. We propose that there is a developmental timing mechanism in embryonic ectoderm that specifies a sequence of competences solely on the basis of the age of the ectoderm.     

A comparative analysis of notochord formation in amphibian embryos]

We studied the origin, structure, and development of the notochord in Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura) embryos. The notochord rudiment is formed in both species at the early gastrula stage as a cluster of polarized chorda-mesoderm cells located along the sagittal plane of the embryo. In Pl. waltlii the notochord rudiment is separated from the gastrocoele roof as a result of contraction of apical cell surfaces. The contraction wave spreads forward and backward along craniocaudal axis, i.e., segregation of the notochord rudiment progresses in two directions simultaneously. Similar process takes place in X. laevis embryos; however, propagation of the contraction wave in the anterior part of the body somewhat differs from that in the posterior part. While the "anterior" contraction wave resembles that in Pl. waltlii embryos, progression of the wave in the posterior part of the body is distinguished by a closer association of the notochord rudiment with ectoderm and the presence of its delamination boundaries with the somite mesoderm.     

Expression pattern of BXR suggests a role for benzoate ligand-mediated signalling in hatching gland function

The Xenopus laevis nuclear receptor BXR has recently been shown to be activated by a class of endogenous benzoate metabolites, indicating the presence of a novel and unsuspected benzoate ligand-dependent signalling pathway. The receptor is expressed ubiquitously in blastula and gastrula stage embryos, and its expression declines during neurula stages. In order to examine further this novel vertebrate signalling system, we have examined the expression of the BXR gene in tailbud stage embryos and adults. We show here that in Xenopus tailbud stage embryos expression is restricted to the hatching gland, suggesting a role in hatching gland function. Neither BXR nor a BXR-VP16 fusion is sufficient to specify hatching gland in neurally-induced tissue. In adults, BXR expression is abundant in the brain and gonads. This expression pattern in adults is distinct from any of the putative mammalian homologues. A nuclear receptor that mediates benzoate signalling has yet to be found in mammals.     

Expression zones of three novel genes abut the developing anterior neural plate of Xenopus embryo

We identified three novel genes that were expressed within the anterior non-neural ectoderm of Xenopus early neurula embryos. The expression of these genes was observed in the different areas complementary to the expression zone of a homeodomain gene Xanf-1 in the anterior neural plate. One of these genes, a Ras-like GTP-ase Ras-dva, marked the anterior placodal ectoderm area; a second, an Agr family homologous gene, XAgr2, was expressed in the anterior-most ectoderm in the cement gland primordium, and a third, novel gene Nlo was expressed in the lateral neural folds. The genes were transiently expressed in the developing cement and hatching gland primordia, and repressed in the mature cement and hatching glands. XAgr2 and Nlo were also expressed in the otic vesicles, and Ras-dva was expressed in the dorso-lateral column of the neural tube.     

A transmembrane protein EIG121L is required for epidermal differentiation during early embryonic development

Epidermal differentiation in the ventral ectoderm of Xenopus embryos is regulated by the bone morphogenetic protein (BMP) pathway. However, it remains unclear how the BMP pathway is activated and induces the epidermal fate in the ventral ectoderm. Here, we identify a novel player in the BMP pathway that is required for epidermal differentiation during Xenopus early embryonic development. We show that Xenopus EIG121L (xEIG121L) protein, an evolutionarily conserved transmembrane protein, is expressed in the ventral ectoderm at the gastrula and neurula stages. Almost complete knockdown of xEIG121L protein with antisense morpholino oligonucleotides in early Xenopus embryos results in severe developmental defects, including the inhibition of epidermal differentiation and the induction of neural genes. Remarkably, our analysis shows that BMP/Smad1 signaling is severely suppressed in the xEIG121L knockdown ectoderm. Moreover, immunoprecipitation and immunostaining experiments suggest that xEIG121L protein physically interacts, and co-localizes, with BMP receptors. Thus, our results identify a novel regulator of the BMP pathway that has a positive role in BMP signaling and plays an essential role in epidermal differentiation during early embryonic development.     

otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation.

We previously showed that otx2 regulates Xenopus cement gland formation in the ectoderm. Here, we show that otx2 is sufficient to direct anterior neural gene expression, and that its activity is required for cement gland and anterior neural determination. otx2 activity at midgastrula activates anterior and prevents expression of posterior and ventral gene expression in whole embryos and ectodermal explants. These data suggest that part of the mechanism by which otx2 promotes anterior determination involves repression of posterior and ventral fates. A dominant negative otx2-engrailed repressor fusion protein (otx2-En) ablates endogenous cement gland formation, and inhibits expression of the mid/hindbrain boundary marker engrailed-2. Ectoderm expressing otx2-En is not able to respond to signals from the mesoderm to form cement gland, and is impaired in its ability to form anterior neural tissue. These results compliment analyses in otx2 mutant mice, indicating a role for otx2 in the ectoderm during anterior neural patterning.     

Spatial and temporal localization of FGF receptors in Xenopus laevis

Summary Mesoderm formation is a result of cell-cell interactions between the vegetal and animal hemisphere and is thought to be mediated by inducing peptide growth factors including members of the FGF and TGF superfamilies. Our immunochemical study analyses the distribution of FGF receptors coded by the human flg gene during embryogenesis of Xenopus laevis. Immunostaining was detected in the dorsal and ventral ectoderm and also in the marginal zone of early cleavage, blastula and gastrula stages. Signals were very strong in the mid and late blastula (stage 8 and 9) and declined slightly in the early gastrula (stage 10). A dramatic decrease was observed up to the late gastrula (stage 11⁺). In stage 13 embryos, immunostaining was only found in cells around the blastopore. Isolated ectoderm cultured in vitro showed a similar temporal expression and decrease of the signal as the normal embryos. These results indicate that receptor expression is independent of the interaction of the animal cells with the vegetal part of the embryo. Of interest is the fact that the signal cannot only be found at or near the cell surface but also within the cell. This suggests the presence of an intracellular isoform of the receptor resulting from the endogenous expression of splice variants and the internalization of transmembrane receptor. Taken together our results suggest that the loss of competence (for bFGF around stage 10) is not directly correlated with the presence of receptors. The possible roles of heparan sulphate glucosaminoglycans (low affinity receptors) and control mechanisms in the intracellular signalling pathway downstream of the receptor level should be taken into consideration.