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The RIKEN Center for Developmental Biology in Kobe, Japan, hosted a meeting entitled 'Regeneration of Organs: Programming and Self-Organization' in March, 2014. Scientists from across the globe met to discuss current research on regeneration, organ morphogenesis and self-organization - and the links between these fields. A diverse range of experimental models and organ systems was presented, and the speakers aptly illustrated the unique power of each. This Meeting Review describes the major advances reported and themes emerging from this exciting meeting., (© 2014. Published by The Company of Biologists Ltd.)

The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucociliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor (Htr3), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the ciliary motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of ciliary beat frequency.

Many organisms and their constituent tissues and organs vary substantially in size but differ little in morphology; they appear to be scaled versions of a common template or pattern. Such scaling involves adjusting the intrinsic scale of spatial patterns of gene expression that are set up during development to the size of the system. Identifying the mechanisms that regulate scaling of patterns at the tissue, organ and organism level during development is a longstanding challenge in biology, but recent molecular-level data and mathematical modeling have shed light on scaling mechanisms in several systems, including Drosophila and Xenopus. Here, we investigate the underlying principles needed for understanding the mechanisms that can produce scale invariance in spatial pattern formation and discuss examples of systems that scale during development.

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.

MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom.

We have identified Xenopus Brain Factor 2 (XBF-2) as a potent neuralizing activity in an expression cloning screen. In ectodermal explants, XBF-2 converts cells from an epidermal to a neural fate. Such explants contain neurons with distinct axonal profiles and express both anterior and posterior central nervous system (CNS) markers. In striking contrast to X-ngnR-1a or X-NeuroD, ectopic expression of XBF-2 in Xenopus embryos results in an expansion of the neural plate to the ventral midline. The enlarged neural plate consists predominantly of undifferentiated neurons. XBF-2 lies downstream of the BMP antagonists noggin, cerberus, and gremlin since ectodermal explants expressing these molecules exhibit strong expression of XBF-2. While XBF-2 does not upregulate the expression of secreted neural inducers, it downregulates the transcription of BMP-4, an epidermal inducer. We show that XBF-2 acts as a transcriptional repressor and that its effects can be phenocopied with either the engrailed or hairy repressor domain fused to the XBF-2 DNA-binding domain. A fusion of the DNA-binding domain to the activator domain of VP16 blocks the effects of XBF-2 and prevents neural plate development in the embryo. This provides evidence that a transcriptional repressor can affect both regional neural development and neurogenesis in vertebrates.

Wnts are secreted signaling molecules implicated in a large number of developmental processes. Frizzled proteins have been identified as likely receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for vertebrate frizzleds has not yet been defined. To assess the endogenous role of frizzled proteins during vertebrate development, we have identified and characterized a Xenopus frizzled gene (xfz8). It is highly expressed in the deep cells of the Spemann organizer prior to dorsal lip formation and in the early involuting marginal zone. Ectopic expression of xfz8 in ventral cells leads to complete secondary axis formation and can synergize with Xwnt-8 while an inhibitory form of xfz8 (Nxfz8) blocks axis duplication by Xwnt-8, consistent with a role for xfz8 in Wnt signal transduction. Expression of Nxfz8 in dorsal cells has profound effects on morphogenesis during gastrulation and neurulation that result in dramatic shortening of the anterior-posterior axis. Our results suggest a role for xfz8 in morphogenesis during the gastrula stage of embryogenesis.

We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

In a differential screen for downstream genes of the neural inducers, we identified two extremely early neural genes induced by Chordin and suppressed by BMP-4: Zic-related-1 (Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2, a Sry-related HMG factor. Expression of the two genes is first detected widely in the prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the proneural gene Xngnr-1, and initiates neural and neuronal differentiation in isolated animal caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2 makes the ectoderm responsive to extracellular signals, demonstrating that the early phase of neural induction involves simultaneous activation of multiple functions.

This review starts from the classical standpoint that there are at least two separable processes acting with respect to axis formation and tissue specification in the early Xenopus embryo: a UV-insensitive event establishing a postgastrula embryo consisting of three concentric germ layers, ectoderm, mesoderm and endoderm, all of a ventral character; and a UV-sensitive event producing tissue of a dorsal type, including somites, notochord and neural tissue, and concomitantly establishing the dorsoventral and anteroposterior axes. The experimental evidence suggesting the molecular basis of the dorsal and ventral pathways is reviewed.

An RNA localized to the vegetal cortex of Xenopus oocytes encodes a novel T-box protein (VegT) capable of inducing either dorsal or posterior ventral mesoderm at different times in development. VegT is a nuclear protein and its C-terminal domain can activate transcription in a yeast reporter assay, observations consistent with VegT functioning as a transcription factor. Zygotic expression is dynamic along the dorsoventral axis, with transcripts first expressed in the dorsal marginal zone. By the end of gastrulation, VegT is expressed exclusively in posterior ventral and lateral mesoderm and is excluded from the notochord. Later expression is confined to a subset of Rohon-Beard cells, a type of primary sensory neuron. In animal cap assays, VegT is capable of converting prospective ectoderm into ventral lateral mesoderm. Such ectopic expression of VegT induces its own expression as well as that of Xwnt-8 in caps, suggesting that a Wnt pathway may be involved. Mis-expression of VegT in dorsal animal blastomeres fated to contribute to brain suppresses head formation. Our results suggest that VegT is a localized transcription factor, which operates sequentially in several developmental pathways during embryogenesis, including dorsoventral and posterior patterning of mesoderm.

The cement gland is a simple secretory organ that marks the anterior-most dorsal ectoderm in Xenopus embryos. In this study, we examine the timing of cement gland induction and the cell interactions that contribute to cement gland formation. Firstly, we show that the outer ectodermal layer, from which the cement gland arises, becomes specified as cement gland by mid-gastrula. Curiously, at early gastrula, the inner layer of the dorsal ectoderm, which does not contribute to the mature cement gland, is strongly and transiently specified as cement gland. Secondly, we show that the mid-gastrula dorsoanterior yolky endoderm, which comes to underlie the cement gland primordium, is a potent inducer of cement gland formation and patterning. The cement gland itself has an anteroposterior pattern, with the gene XA expressed only posteriorly. Dorsoanterior yolky endoderm greatly enhances formation of large, patterned cement glands in partially induced anterodorsal ectoderm, but is unable to induce cement gland in naive animal caps. Neural tissue is induced less frequently than cement gland by the dorsoanterior yolky endoderm, suggesting that the endoderm induces cement gland directly. Thirdly, we demonstrate that the ventral ectoderm adjacent to the cement gland attenuates cement gland differentiation late during gastrulation. The more distant ventral mesendoderm is also a potent inhibitor of cement gland formation. These are the first data showing that normal ventral tissues can inhibit cement gland differentiation and suggest that cement gland size and position may be partly regulated by negative signals. Previous work has shown that cement gland can be induced by neural plate and by dorsal mesoderm. Together, these data suggest that cement gland induction is a complex process regulated by multiple positive and negative cell interactions.

We have isolated and characterized Xenopus cDNA clones for a new transcription factor that represents an early marker for the developing heart. The cDNAs encode a protein that we have designated GATA-4; it contains the highly conserved DNA-binding domain that characterizes this family of cell-type restricted transcriptional activators. Whole-embryo in situ analysis of Xenopus embryos demonstrates that the GATA-4 gene is transcribed in presumptive cardiac ventral mesoderm at the time that bilateral progenitors fuse and form the cardiac tube. GATA-4 is therefore the earliest molecular marker of cardiogenesis yet characterized. By stage 30, the GATA-4 mRNA is expressed in the developing atria and ventricles; at stage 38, cross-sections reveal that the gene is active in the endocardial layer, but not in myocardium. By stage 40, GATA-4 message is detected in the great vessels. In the adult frog, the GATA-4 gene is highly transcribed in heart and gut; lower levels of message are detected in various endoderm-derived tissues and gonads. Expression in the stomach is largely confined to the epithelium. The GATA-4 gene is first activated at stage 11; mRNA is initially present throughout the marginal zone of explants and later partially localized to the ventral marginal zone. GATA-4 mRNA is also detected at high levels in cultured endodermal explants derived from the vegetal region of early embryos. In mesoderm induction experiments, GATA-4 transcription is not induced in animal caps treated with activin or bFGF. The GATA-4 gene may provide a new early marker for studying the inductive processes that lead to the formation of the cardiovascular system and for the specification of the endocardial lineage.

The isthmic nucleus (IN) is a visual relay centre of the frog brain. It receives afferent projection from the optic tectum of the same side and projects bilaterally to both tecta. In young postmetamorphic Xenopus frogs, the survival of neurones in the IN on both sides was studied following the complete removal of the right tectum. In 6- to 8-week-old frogs, the right tectum was surgically removed and the operated animals allowed to survive for 1 to 13 weeks after operation. In selected animals, 3 days before the intended sacrifice, the postoptic commissure was transected and the cut isthmotectal fibres filled with horseradish peroxidase (HRP). In serial paraffin sections of the midbrain, the numbers of surviving and dying (pyknotic) neurones in the left and right IN were counted. The soma size of viable isthmic neurones and the volume of both IN were measured. Pyknotic neurones were seen between 1 and 6 weeks after operation in both the left and right IN, although the rate of cell loss was much greater in the latter. Virtually all the neurones of the right IN degenerated by 6 weeks after tectal ablation. In contrast, approximately 60% of neurones of the left IN survived. HRP histochemistry showed labelled isthmic neurones both in the left and right IN up to 3 weeks after operation. Thereafter, HRP-labelled neurones appeared only in the left IN. These observations indicate that the removal of the natural target of isthmic neurones brings about severe neurone death.(ABSTRACT TRUNCATED AT 250 WORDS)