Neuronatin Promotes Neural Lineage in ESCs via Ca2+ Signaling

Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underling neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca2+ ([Ca2+]i) by antagonizing Ca2+-ATPase isoform 2 (sarco/endoplasmic reticulum Ca2+-ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway. STEM CELLS 2010;28:1950–196

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

More Than 1,001 Problems with Protein Domain Databases: Transmembrane Regions, Signal Peptides and the Issue of Sequence Homology

Large-scale genome sequencing gained general importance for life science because functional annotation of otherwise experimentally uncharacterized sequences is made possible by the theory of biomolecular sequence homology. Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. Having the same fold imposes strict conditions over the packing in the hydrophobic core requiring similarity of hydrophobic patterns. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Thus, matching of SPs/TMs creates the illusion of matching hydrophobic cores. Therefore, inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment-mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, we show explicit examples that the scores of clearly false-positive hits, even in global-mode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, we find that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, false-positive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. We suggest a workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users

Regulated Fluctuations in Nanog Expression Mediate Cell Fate Decisions in Embryonic Stem Cells

The notion that the differentiated state of a cell population is determined simply by expression of specific marker genes is changing. In this work, the authors reveal that a pluripotent cell population comprises cells with temporal fluctuations in the expression of Nanog

Deciphering the stem cell machinery as a basis for understanding the molecular mechanism underlying reprogramming

Stem cells provide fascinating prospects for biomedical applications by combining the ability to renew themselves and to differentiate into specialized cell types. Since the first isolation of embryonic stem (ES) cells about 30 years ago, there has been a series of groundbreaking discoveries that have the potential to revolutionize modern life science. For a long time, embryos or germ cell-derived cells were thought to be the only source of pluripotency—a dogma that has been challenged during the last decade. Several findings revealed that cell differentiation from (stem) cells to mature cells is not in fact an irreversible process. The molecular mechanism underlying cellular reprogramming is poorly understood thus far. Identifying how pluripotency maintenance takes place in ES cells can help us to understand how pluripotency induction is regulated. Here, we review recent advances in the field of stem cell regulation focusing on key transcription factors and their functional interplay with non-coding RNAs

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