Expression and Roles of Teneurins in Zebrafish

The teneurins, also known as Ten-m/Odz, are highly conserved type II transmembrane glycoproteins widely expressed throughout the nervous system. Functioning as dimers, these large cell-surface adhesion proteins play a key role in regulating neurodevelopmental processes such as axon targeting, synaptogenesis and neuronal wiring. Synaptic specificity is driven by molecular interactions, which can occur either in a trans-homophilic manner between teneurins or through a trans-heterophilic interaction across the synaptic cleft between teneurins and other cell-adhesion molecules, such as latrophilins. The significance of teneurins interactions during development is reflected in the widespread expression pattern of the four existing paralogs across interconnected regions of the nervous system, which we demonstrate here via in situ hybridization and the generation of transgenic BAC reporter lines in zebrafish. Focusing on the visual system, we will also highlight the recent developments that have been made in furthering our understanding of teneurin interactions and their functionality, including the instructive role of teneurin-3 in specifying the functional wiring of distinct amacrine and retinal ganglion cells in the vertebrate visual system underlying a particular functionality. Based on the distinct expression pattern of all teneurins in different retinal cells, it is conceivable that the combination of different teneurins is crucial for the generation of discrete visual circuits. Finally, mutations in all four human teneurin genes have been linked to several types of neurodevelopmental disorders. The opportunity therefore arises that findings about the roles of zebrafish teneurins or their orthologs in other species shed light on the molecular mechanisms in the etiology of such human disorders

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

A gene regulatory network for neural induction

During early vertebrate development, signals from a special region of the embryo, the organizer, can re-direct the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here we undertake a comprehensive analysis, in very fine time-course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen's node). Using transcriptomics and epigenomics we generate a Gene Regulatory Network comprising 175 transcriptional regulators and 5,614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing and reporter assays we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates

Expression of <i>Asterix</i> during development (continued).

<p>Sections through embryos at stages 4+−18, at the levels indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019157#pone-0019157-g003" target="_blank">Fig. 3</a>. Q shows a coronal section through an embryo at stage 16, showing expression in the notochord.</p

Molecular characterization of Obelix.

<p><b>A.</b> Sequence alignment of Obelix protein (AY103477) with ESTs for EIF1A-related proteins from several species. Residues are displayed in different colours based on different aminoacid families and degree of homology is represented by conservation of these sites. Conserved OB-like domain is shown as a block in the alignment. Species are abbreviated as follows: ag, <i>Anopheles</i> mosquito (BM594550); bt, cow (BF043073); ce, <i>C. elegans</i> (AV203381); ci, <i>Ciona</i> (AV841463); dm, <i>Drosophila melanogaster</i> (BE977318); dr, zebrafish (BM859434); hs, human (BG149615); mm, mouse (BI103120); ss, pig (BG610103); rn, rat (BF420639); xl, <i>Xenopus laevis</i> (BG730245); xt, <i>Xenopus tropicalis</i> (AL637659). <b>B.</b> Phylogenetic tree with bootstrap values comparing the full-length sequences of Obelix in a variety of species, showing that eIF1A and Obelix segregate into two distinct sub-classes of OB-containing proteins. The LG model was used to construct the tree and bootstrap values were calculated from 1000 replicates.</p

Regulation of <i>Obelix</i> by various secreted factors.

<p><b>A–P</b>. The ability of various peptide factors to induce <i>Obelix</i> expression was tested by local application of beads soaked in the protein or pellets of COS-1 cells transfected with a construct encoding the factor into the area opaca of a host embryo (A). Examples of FGF4 beads (B), Chordin (C) and Noggin (D) cells, FGF8/control beads (E), Dickkopf (F), Cerberus (G) cells and HGF/SF beads (H) are shown. I–P show sections through the grafted region of the embryos in B–H at the levels indicated. <b>Q–U</b>. Co-transplantation of a quail Hensen's node with beads soaked in the FGF inhibitor SU5402 has little or no effect: Obelix is still induced (Q–U). Q shows a grafted embryo fixed after 6 hours, and R is an example of an embryo grown overnight after the graft. S–U are sections through these embryos at the levels indicated in Q and R. Quail cells are stained with QCPN (brown). Note that some probes attach non-specifically to some types of beads and to COS cell pellets (eg. panels K–H).</p

Expression of <i>Asterix</i> during development.

<p>Embryos at stages XI (A), XII (B), 3 (C), 4+ (D), 6 (E), 7 (F), 9 (G), 10 (H), 11 (I), 14 (J), 16 (K), 17 (L) and 18 (M) are shown. The horizontal lines and letters refer to the levels at which sections in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019157#pone-0019157-g004" target="_blank">Fig. 4</a> were taken.</p

Obelix is intracellular and localizes to the nucleus.

<p><b>A.</b> Obelix protein can be retrieved from cell extracts (C) but not from the supernatant (S) of transfected COS-1 cells, and detected by Western blotting. <b>B–D.</b> Nuclear localization of Myc-tagged Obelix protein can be seen in transfected COS-1 cells (B, C) as well as in the neural plate of a chick embryo (D). In B and D the anti-Myc antibody is revealed by peroxidase staining with diaminobenzidine; in C the signal is revealed with Cy3-coupled anti-mouse antibody.</p

Time-course of markers during neural induction and their regulation by signals.

<p>Temporal hierarchy of deployment of 13 early neural markers. The colored lines at the bottom of the figure represent the period of expression of these genes, in relation to the time at which they are induced following a graft of Hensen's node into the area opaca (in hours on the scale above) and in relation to the stage of normal embryos at which they are expressed (stage shown above the time line). The diagrams above these stages schematize the domains of expression. The genes fall into three “epochs”: those induced by a node within 3 hours start to be expressed in normal embryos before streak formation (red). Those induced by a node in 4–5 hours begin their expression at the mid- to late-primitive streak stage (blue) and those that are induced by a node at 12–13 hours do not begin their expression until the end of gastrulation, in the forming neural plate (green).</p

<i>Obelix</i> expression during early development.

<p>Expression of <i>Obelix</i> by in situ hybridization at stages 3+ (A), 5 (B), 7 (C) and 11 (D). E–G are sections through the levels shown in A–C. Expression is localized in the neural plate, neural tube and their derivatives.</p