A database of microRNA expression patterns in Xenopus laevis

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase

Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-β Antagonist, Controls Early Neuroectoderm Specification in Xenopus

Xenopus maternal Norrin, which activates Wnt signaling but inhibits TGF-β family molecules, is essential for neuroectoderm formation. Loss of TGF-β inhibition in Norrin may contribute to the development of Norrie disease

Calfacilitin is a calcium channel modulator essential for initiation of neural plate development.

Calcium fluxes have been implicated in the specification of the vertebrate embryonic nervous system for some time, but how these fluxes are regulated and how they relate to the rest of the neural induction cascade is unknown. Here we describe Calfacilitin, a transmembrane calcium channel facilitator that increases calcium flux by generating a larger window current and slowing inactivation of the L-type CaV1.2 channel. Calfacilitin binds to this channel and is co-expressed with it in the embryo. Regulation of intracellular calcium by Calfacilitin is required for expression of the neural plate specifiers Geminin and Sox2 and for neural plate formation. Loss-of-function of Calfacilitin can be rescued by ionomycin, which increases intracellular calcium. Our results elucidate the role of calcium fluxes in early neural development and uncover a new factor in the modulation of calcium signalling

Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

BACKGROUND: It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. METHODS AND PRINCIPAL FINDINGS: Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells

CRIM1 Complexes with ß-catenin and Cadherins, Stabilizes Cell-Cell Junctions and Is Critical for Neural Morphogenesis

In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions

The Function of Heterodimeric AP-1 Comprised of c-Jun and c-Fos in Activin Mediated Spemann Organizer Gene Expression

BACKGROUND:Activator protein-1 (AP-1) is a mediator of BMP or FGF signaling during Xenopus embryogenesis. However, specific role of AP-1 in activin signaling has not been elucidated during vertebrate development. METHODOLOGY/PRINCIPAL FINDINGS:We provide new evidence showing that overexpression of heterodimeric AP-1 comprised of c-jun and c-fos (AP-1(c-Jun/c-Fos)) induces the expression of BMP-antagonizing organizer genes (noggin, chordin and goosecoid) that were normally expressed by high dose of activin. AP-1(c-Jun/c-Fos) enhanced the promoter activities of organizer genes but reduced that of PV.1, a BMP4-response gene. A loss of function study clearly demonstrated that AP-1(c-Jun/c-Fos) is required for the activin-induced organizer and neural gene expression. Moreover, physical interaction of AP-1(c-Jun/c-Fos) and Smad3 cooperatively enhanced the transcriptional activity of goosecoid via direct binding on this promoter. Interestingly, Smad3 mutants at c-Jun binding site failed in regulation of organizer genes, indicating that these physical interactions are specifically necessary for the expression of organizer genes. CONCLUSIONS/SIGNIFICANCE:AP-1(c-Jun/c-Fos) plays a specific role in organizer gene expression in downstream of activin signal during early Xenopus embryogenesis

Distinct Steps of Neural Induction Revealed by Asterix, Obelix and TrkC, Genes Induced by Different Signals from the Organizer

The amniote organizer (Hensen's node) can induce a complete nervous system when grafted into a peripheral region of a host embryo. Although BMP inhibition has been implicated in neural induction, non-neural cells cannot respond to BMP antagonists unless previously exposed to a node graft for at least 5 hours before BMP inhibitors. To define signals and responses during the first 5 hours of node signals, a differential screen was conducted. Here we describe three early response genes: two of them, Asterix and Obelix, encode previously undescribed proteins of unknown function but Obelix appears to be a nuclear RNA-binding protein. The third is TrkC, a neurotrophin receptor. All three genes are induced by a node graft within 4–5 hours but they differ in the extent to which they are inducible by FGF: FGF is both necessary and sufficient to induce Asterix, sufficient but not necessary to induce Obelix and neither sufficient nor necessary for induction of TrkC. These genes are also not induced by retinoic acid, Noggin, Chordin, Dkk1, Cerberus, HGF/SF, Somatostatin or ionomycin-mediated Calcium entry. Comparison of the expression and regulation of these genes with other early neural markers reveals three distinct “epochs”, or temporal waves, of gene expression accompanying neural induction by a grafted organizer, which are mirrored by specific stages of normal neural plate development. The results are consistent with neural induction being a cascade of responses elicited by different signals, culminating in the formation of a patterned nervous system

Role of BMP, FGF, Calcium Signaling, and Zic Proteins in Vertebrate Neuroectodermal Differentiation

More than a decade has passed since Zic family zinc finger proteins were discovered to be transcription factors controlling neuroectodermal differentiation (neural induction) in Xenopus laevis embryos. Although BMP-signal blocking has been shown to be a major upregulator of Zic genes in neuroectodermal differentiation, recent studies have revealed that FGF signaling and intracellular calcium elevation are also involved in regulating the expression of Zic genes. Different regulatory mechanisms have been found for the Zic1 and Zic3 genes, raising the possibility that functional synergism between them partly accounts for the integration of BMP-signal blocking and FGF signaling in neuroectodermal differentiation. Furthermore, mammalian Zic1 and Zic3 have been found to be neural-cell-fate-inducing and pluripotency-maintaining factors, respectively, leading us to the intriguing question of whether the mechanism underlying amphibian neuroectodermal differentiation is applicable to mammals. Comprehensive understanding of the Zic family genes is therefore essential for the study of the neuroectodermal differentiation and stem cell biology

Bmp4 Is Essential for the Formation of the Vestibular Apparatus that Detects Angular Head Movements

Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-β), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification