Progressive determination during formation of the anteroposterior axis in Xenopus laevis

The cement gland is an ectodermal organ in the head of frog embryos, lying anterior to any neural tissue. As analyzed by specific RNA expression, cement gland, like neural tissue, was induced by the dorsal mesoderm. Interestingly, mesoderm with the highest cement gland-inducing potential lay posterior to the ectoderm fated to form this organ, indicating that its induction occurred at a distance from the inducer source. Cement gland induction first occurred during early gastrulation. However, most initially induced cells did not contribute to the mature cement gland, but instead formed part of the neural plate. This change in fate could be reconstituted in vitro. These results suggest that determination of part of the anteroposterior axis occurs progressively, where future neural ectoderm is first induced to a cement glandlike state. As gastrulation proceeds, further induction by mesoderm may override this state, which persists only in the extreme anterior of the embryo.     

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.     

Zebrafish mdk2, a novel secreted midkine, participates in posterior neurogenesis

Patterning the neural plate in vertebrates depends on complex interactions between a variety of secreted growth factors. Here we describe a novel secreted factor in zebrafish, named mdk2, related to the midkine family of heparin-binding growth factors that is involved in posterior neural development. mdk2 is expressed shortly after the onset of gastrulation in the presumptive neural plate cells of the epiblast, and this expression is enhanced by exogenous retinoic acid. Ectopic expression of mdk2 enhances neural crest cell fates at the lateral edges of the caudal neural plate, concomitant with a repression of anterior structures and mesendodermal and ectodermal markers. Reciprocally, ectopic expression of a dominant negative mdk2 results in severe deficiencies of structures posterior to the midbrain-hindbrain boundary, with negligible effects on anterior structures. In these embryos, the expression of hindbrain and neural crest markers is strongly reduced, and the formation of posterior primary moto- and sensory neurons is blocked. Analyses in mutant zebrafish embryos shows that expression of mdk2 is independent of FGF8 and nodal-related-1 signaling, but is under negative control of BMP signaling. These data support the hypothesis that mdk2 participates in posterior neural development in zebrafish.     

Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity

In Xenopus, ectodermal patterning depends on a mediolateral gradient of BMP signaling, higher in the epidermis and lower in the neuroectoderm. Neural crest cells are specified at the border between the neural plate and the epidermis, at intermediate levels of BMP signaling. We recently described a novel secreted protein, Tsukushi (TSK), which works as a BMP antagonist during chick gastrulation. Here, we report on the Xenopus TSK gene (X-TSK), and show that it is involved in neural crest specification. X-TSK expression accumulates after gastrulation at the anterior-lateral edges of the neural plate, including the presumptive neural crest region. In gain-of-function experiments, X-TSK can strongly enhance neural crest specification by the dorsolateral mesoderm or X-Wnt8 in ectodermal explants, while the electroporation of X-TSK mRNA in the lateral ectoderm of embryos after gastrulation can induce the expression of neural crest markers in vivo. By contrast, depletion of X-TSK in explants or embryos impairs neural crest specification. Similarly to its chick homolog, X-TSK works as a BMP antagonist by direct binding to BMP4. However, X-TSK can also indirectly regulate BMP4 mRNA expression at the neural plate border via modulation of the Delta-Notch signaling pathway. We show that X-TSK directly binds to the extracellular region of X-delta-1, and modulates Delta-dependent Notch activity. We propose that X-TSK plays a key role in neural crest formation by directly regulating BMP and Delta activities at the boundary between the neural and the non-neural ectoderm.     

Xenopus Tetraspanin-1 regulates gastrulation movements and neural differentiation in the early Xenopus embryo

The tetraspanin family of four-pass transmembrane proteins has been implicated in fundamental biological processes, including cell adhesion, migration, and proliferation. Tetraspanins interact with various transmembrane proteins, establishing a network of large multimolecular complexes that allows specific lateral secondary interactions. Here we report the identification and functional characterization of Xenopus Tetraspanin-1 (xTspan-1). At gastrula and neurula, xTspan-1 is expressed in the dorsal ectoderm and neural plate, respectively, and in the hatching gland, cement gland, and posterior neural tube at tailbud stages. The expression of xTspan-1 in the early embryo is negatively regulated by bone morphogenetic protein (BMP) and stimulated by Notch signals. Microinjection of xTspan-1 mRNA interfered with gastrulation movements and reduced ectodermal cell adhesion in a cadherin-dependent manner. Morpholino knock-down of endogenous xTspan-1 protein revealed a requirement of xTspan-1 for gastrulation movements and primary neurogenesis. Our data suggest that xTspan-1 could act as a molecular link between BMP signalling and the regulation of cellular interactions that are required for gastrulation movements and neural differentiation in the early Xenopus embryo.     

BRAIN INDUCTION IN VARIOUSLY AGED PRESUMPTIVE ECTODERMS BY HEAD ORGANIZER IN CYNOPS PYRRHOGASTER

Brain formation in variously aged presumptive ectoderms of Cynops pyrrhogaster under the influence of the head organizer was examined by the sandwich method. The head organizer was obtained from the middle portion of the archenteron roof at the slit-blastopore stage. The presumptive ectoderm was taken from 0- to 36-hr exogastrulae. Exogastrulae were prepared from the earliest gastrulae just before invagination (0-hr embryos). The presumptive neural plate overlying the archenteron roof used as organizer was cultivated in an envelope of belly ectoderm from an early neurula.
The following results were obtained: 1) Brain induction was almost entirely restricted to explants covered with 6-hr ectoderm and its frequency was low. 2) The presumptive neural plate above the head organizer was almost completely determined as neural tissues. 3) The head organizer showed a tendency to differentiate into more endodermal and less mesodermal tissues than those expected from its prospective fate.
Brain induction in normal development and the relationship between neural tissue formation in variously aged presumptive ectoderms and the time necessary for neural induction are discussed.     

Identification and characterization of Xenopus kctd15, an ectodermal gene repressed by the FGF pathway

The FGF pathway regulates a variety of developmental processes in animals through activation and/or repression of numerous target genes. Here we have identified a Xenopus homolog of potassium channel tetramerization domain containing 15 (KCTD15) as an FGF-repressed gene. Kctd15 expression is first detected at the gastrula stage and gradually increases until the tadpole stage. Whole-mount in situ hybridization reveals that the spatial expression of kctd15 is tightly regulated during early embryogenesis. While kctd15 is uniformly expressed throughout the presumptive ectoderm at the early gastrula stage, its expression becomes restricted to the non-neural ectoderm and is excluded from the neural plate at the early neurula stage. At the mid-neurula stage, kctd15 shows a more restricted distribution pattern in regions that are located at the anterior, lateral or medial edge of the neural fold, including the preplacodal ectoderm, the craniofacial neural crest and the prospective roof plate. At the tailbud stage, kctd15 expression is mainly detected in neural crest- or placode-derived tissues that are located around the eye, including the mandibular arch, trigeminal ganglia and the olfactory placode. FGF represses kctd15 expression in ectodermal explants, and the inhibition of FGF receptor with a chemical compound dramatically expands the region expressing kctd15 in whole embryos. Dorsal depletion of kctd15 in Xenopus embryos leads to bent axes with reduced head structures, defective eyes and abnormal somites, while ventral depletion causes defects in ventral and caudal morphologies. These results suggest that kctd15 is an FGF-repressed ectodermal gene required for both dorsal and ventral development.     

Regulation of ectodermal differentiation in Xenopus laevis animal caps treated with TPA and ammonium chloride

Animal caps isolated from Xenopus laevis embryos at the blastula stage were treated sequentially with NH₄Cl, a known cement gland inducer, and with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a known neural inducer. The two artificial inducers were also used in reverse order to see if they can mimic the natural inducers acting during the progressive determination of the ectodermal organ. Immunofluorescence and whole-mount in situ hybridization were used to study the expression of tubulin, taken to indicate an early step on the pathway of cell elongation, and neural cell adhesion molecule (N-CAM) taken to indicate an early step in the determination of the nervous system. The expression of XCG-1, a marker of early specification of the cement gland, was also studied. The results showed that the two artificial inducers can mimic the effects of the natural inducers in animal cap explants. The TPA behaves like a neural inducer, reducing the number and the extension of the cement gland when added to the medium in addition to NH₄Cl, before or after NH₄Cl treatment. In the process of cement gland/neural induction, it is possible to redirect the ectoderm already specified as cement gland to neural tissue, but it does not seem possible to respecify the neural tissue as cement gland. Moreover, the animal caps were also cut into dorsal and ventral parts and the two halves were treated separately. The results were similar to those obtained with treatment of the entire animal cap, suggesting that a dorsal-ventral pattern is not yet established before the gastrula stage, and that in normal embryos there are boundaries between the effects of different inducers.     

A novel fork head gene mediates early steps during Xenopus lens formation.

Xlens1 is a novel Xenopus member of the fork head gene family, named for its nearly restricted expression in the anterior ectodermal placode, presumptive lens ectoderm (PLE), and anterior epithelium of the differentiated lens. The temporal and spatial restriction of its expression suggests that: (1) Xlens1 is transcribed initially at neural plate stages in response to putative signals from the anterior neural plate that transform lens-competent ectoderm to lens-biased ectoderm; (2) further steps in the process of lens-forming bias restrict Xlens1 expression to the presumptive lens ectoderm (PLE) during later neural plate stages; (3) interactions with the optic vesicle maintain Xlens1 expression in the lens placode; and (4) Xlens1 expression is downregulated as committed lens cells undergo terminal differentiation. Induction assays demonstrate that pax6 induces Xlens1 expression, but unlike pax6, Xlens1 cannot induce the expression of the lens differentiation marker beta-crystallin. In the whole embryo, overexpression of Xlens1 in the lens ectoderm causes it to thicken and maintain gene expression characteristics of the PLE. Also, this overexpression suppresses differentiation in the lens ectoderm, suggesting that Xlens1 functions to maintain specified lens ectoderm in an undifferentiated state. Misexpression of Xlens1 in other regions causes hypertrophy of restricted tissues but only occasionally leads ectopic sites of gamma-crystallin protein expression in select anterior head regions. These results indicate that Xlens1 expression alone does not specify lens ectoderm. Lens specification and differentiation likely depends on a combination of other gene products and an appropriate level of Xlens1 activity.     

Tissue-specific expression of an Ornithine decarboxylase paralogue, XODC2, in Xenopus laevis

Ornithine decarboxylase (ODC) is involved in the biosynthesis of polyamines and hence has been found in almost all types of cells studied. Therefore it is frequently used as internal standard. We isolated a cDNA, XODC2, which is a paralogue to ubiquitous ODC and expressed in a spatial and temporal manner during the early embryogenesis of Xenopus laevis. Expression of XODC2was first detected at the animal pole at stage 9. During neurula stages the signals were found both in the extreme anterior and posterior part of the dorsal body axis. In tailbud stages the expression is further shifted to both the tail and head areas and gradually restricted to distinct tissues: forebrain, inner layer of epidermis of the head area, stomodeal-hypophyseal anlage, frontal gland, ear vesicle, branchial arches, the front tip of neural tube and proctodeum. In addition, signals were also found in the inner layer of epidermis underneath the cement gland during early tailbud stages while in later tailbud stages signals were detected at the apical zone of the cement gland. Comparative studies indeed could confirm that XODC1 in contrast to XODC2 is expressed ubiquitously throughout the whole embryos during early development of Xenopus laevis.     

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.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.