A Requirement for Zic2 in the Regulation of Nodal Expression Underlies the Establishment of Left-Sided Identity

ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution

B1 SOX Coordinate Cell Specification with Patterning and Morphogenesis in the Early Zebrafish Embryo

The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization, RT–PCR, and microarray analyses. Importantly, these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: (1) early dorsoventral patterning by controlling bmp2b/7; (2) gastrulation movements via the regulation of pcdh18a/18b and wnt11, a non-canonical Wnt ligand gene; (3) neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively) and ascl1a (negatively), and regional transcription factor genes, e.g., hesx1, zic1, and rx3; and (4) neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX. We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo

The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue

<p>Abstract</p> <p>Background</p> <p>Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized <it>in vitro </it>and <it>in vivo </it>the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells.</p> <p>Results</p> <p>We show that <it>Ens-1 </it>LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the <it>Ens-1 </it>gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of <it>Ens-1</it>.</p> <p>Conclusion</p> <p>Our results show that <it>Ens-1 </it>LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, <it>Ens-1 </it>LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.</p

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

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

Immune activation by life-shortening Wolbachia and reduced filarial competence in mosquitoes

Wolbachia strain wMelPop reduces the longevity of its Drosophila melanogaster host and, when introduced into the mosquito Aedes aegypti, halves its life span. We show that wMelPop induces up-regulation of the mosquito’s innate immune system and that its presence inhibits the development of filarial nematodes in the mosquito. These data suggest that wMelPop could be used in the global effort to eliminate lymphatic filariasis and possibly for the control of other mosquito-borne parasites where immune preactivation inhibits their development. The cost of constitutive immune up-regulation may contribute to the life-shortening phenotype

Wolbachia strain wMel induces cytoplasmic incompatibility and blocks dengue transmission in Aedes albopictus

Wolbachia inherited bacteria are able to invade insect populations using cytoplasmic incompatibility and provide new strategies for controlling mosquito-borne tropical diseases, such as dengue. The overreplicating wMelPop strain was recently shown to strongly inhibit the replication of dengue virus when introduced into Aedes aegypti mosquitoes, as well as to stimulate chronic immune up-regulation. Here we show that stable introduction of the wMel strain of Drosophila melanogaster into Aedes albopictus, a vector of dengue and other arboviruses, abolished the transmission capacity of dengue virus-challenged mosquitoes. Immune up-regulation was observed in the transinfected line, but at a much lower level than that previously found for transinfected Ae. aegypti. Transient infection experiments suggest that this difference is related to Ae. albopictus immunotolerance of Wolbachia, rather than to the Wolbachia strain used. This study provides an example of strong pathogen inhibition in a naturally Wolbachia-infected mosquito species, demonstrating that this inhibition is not limited to naturally naïve species, and suggests that the Wolbachia strain is more important than host background for viral inhibition. Complete bidirectional cytoplasmic incompatibility was observed with WT strains infected with the naturally occurring Ae. albopictus Wolbachia, and this provides a mechanism for introducing wMel into natural populations of this species