Nodal signaling is required for closure of the anterior neural tube in zebrafish

Background: Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish

Molecular Cloning and Pharmacological Characterization of Two Novel GnRH Receptors in the Lamprey (Petromyzon marinus)

This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (IGnRH-R-2 and IGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands IGnRH-II and -III induced an inositol phosphate (IP) response at IGnRH-R-2 and IGnRH-R-3, whereas the ligand IGnRH-I did not stimulate an IP response. IGnRH-II was a more potent activator of lGnRH-R-3 than IGnRH-II. Stimulation of IGnRH-IIR-2 and IGnRH-R-3 testing all three IGnRH ligands did not elicit a cAMP response. IGnRH-R-2 has a higher binding affinity in response to IGnRH-R-2 than IGnRH-R-2, whereas IGnRH-R-3 has a higher binding affinity in response to IGnRH-R-2 than IGnRH-III. IGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that IGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and IGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate. (Endocrinology 153: 3345-3356, 2012

Nodal signaling is required for closure of the anterior neural tube in zebrafish-4

<p><b>Copyright information:</b></p><p>Taken from "Nodal signaling is required for closure of the anterior neural tube in zebrafish"</p><p>http://www.biomedcentral.com/1471-213X/7/126</p><p>BMC Developmental Biology 2007;7():126-126.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2214732.</p><p></p>tal sections though the anterior neural keel with anterior to the left and dorsal to the top. (C-D') Parasagittal sections with anterior to the left and dorsal to the top. Panels C' and D' are higher magnifications of the boxed regions in panels C and D, respectively. Arrows point to N-cad labeling in the cell membrane, while the arrowhead indicates labeling in the cytoplasm. (E, F) Transverse sections through the anterior neural tube, dorsal to the top. n ≥ 30 embryos for each sample. Embryos are at 95–100% epiboly (95–100%) or the 5–6 somite stage (5–6 s). Scale bars: 40 μm (C, D, E, F), 20 μm (A, B, C', D')

Nodal signaling is required for closure of the anterior neural tube in zebrafish-7

<p><b>Copyright information:</b></p><p>Taken from "Nodal signaling is required for closure of the anterior neural tube in zebrafish"</p><p>http://www.biomedcentral.com/1471-213X/7/126</p><p>BMC Developmental Biology 2007;7():126-126.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2214732.</p><p></p>6 cell stage, raised until 24 hpf, and imaged (A-C') live by (A-C) bright field and (A'-C') fluorescent microscopy, and then (D-H") fixed and processed for whole mount in situ hybridization with to mark the pineal, to mark the hatching glands (hg), and to mark the notochord (nc). Panels with the same letter are different views of the same embryo. In addition, the embryos shown in the A, B, and C rows are the same embryos as in the D, F, and G rows, as indicated by the black arrows. (A'-C') Green arrows indicate fluorescence in the anterior mesendoderm, green arrowheads fluorescence in the notochord. (D-H) Closed arrowheads indicate the pineal organ, and open arrowheads the notochord. Whether an embryo is positive (+) or negative (-) for notochord staining is indicated. (D'-H') Open arrowheads point to hatching gland cells, and whether the embryo is positive or negative for these cells is indicated as in D-H'. (D"-H") Closed arrowheads point to the regions of pineal precursors, and the morphology of the pineal anlage is noted. (A-H') Sagittal views with anterior to the left. (D"-H") Dorsal views of the back of the head. Scale bars: 100 μm (A-H), 50 μm (D'-H")

Nodal signaling is required for closure of the anterior neural tube in zebrafish-2

<p><b>Copyright information:</b></p><p>Taken from "Nodal signaling is required for closure of the anterior neural tube in zebrafish"</p><p>http://www.biomedcentral.com/1471-213X/7/126</p><p>BMC Developmental Biology 2007;7():126-126.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2214732.</p><p></p> head of the WT embryo is smooth and rounded, (B) the head of the embryo is pointed (arrow). (C-E) Higher magnification of the anterior embryo reveals variability in the brain morphology of mutants. In (C) WT embryos and (D) some mutants, the border between the tectum and tegmentum (open arrowheads) appears as a smooth, straight line. (E) However, in some mutants the border appears to be abnormally shaped or indistinct (open arrowhead), suggesting that the morphology of tectum or tegmentum is perturbed. (F-J) Embryos were fixed at 1 dpf, and processed for in situ hybridization with antisense probes for the pineal gene and the dorsal neural tube gene . In (F) WT and (G) embryos with a single, round pineal anlage, the expressing cells (open arrowheads) form a single domain along the dorsal neural tube. In contrast, embryos with an (H) elongated or (I-J) divided pineal anlage have two parallel lines of expressing cells. (K-P) Embryos were fixed at 1 dpf, processed for in situ hybridization with an antisense probe for , and then either (K-N) imaged in dorsal view, anterior to the left or (O, P) cut through -expressing rhombomere 5 to bisect the embryo into anterior and posterior halves. The locations of the otic vesicles (o), rhombomere 5 (arrows), and midline (open arrowhead) are indicated. A potential region of midline is marked by the open arrowhead in P. (Q, R) 14 μM frozen cross sections through the diencephalon of 1 dpf (Q) WT or (R) MZembryos stained for expression. The midline of the brain (open arrowhead), and pineal precursors (closed arrowheads) are indicated. Dotted lines outline the neural tubes in panels O-R. Scale bars: 200 μm (A,B), 100 μm (C-E), 30 μm (F-J), 50 μm (K-R)

Nodal signaling is required for closure of the anterior neural tube in zebrafish-8

<p><b>Copyright information:</b></p><p>Taken from "Nodal signaling is required for closure of the anterior neural tube in zebrafish"</p><p>http://www.biomedcentral.com/1471-213X/7/126</p><p>BMC Developmental Biology 2007;7():126-126.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2214732.</p><p></p>isense probe for the gene , which is expressed in the pineal precursors (arrowheads). Images are dorsal views of the entire embryo, anterior to the top. (A) At the 2–3 somite stage, pineal precursors are located in two widely spaced lateral domains. (B) By the 5–6 somite stage, these domains have moved towards the dorsal midline of the forebrain. (C) By the 7–8 somite stage, a single, round-shaped pineal anlage has formed. (D-F) At 2 dpf, mutant embryos from the same clutch have a wide range of eye phenotypes. Frontal views of live embryos with dorsal to the top. (D) The eyes of WT embryos and some mutants (not shown) are completely separated from one another. (E) Other mutants have partially fused eyes that form two lenses or (F) a single eye with one lens. (G-L) Embryos at the (G-I) 7–8 somite stage and (J-L) 24 hpf were processed for whole mount in situ hybridization with a probe for the pineal gene (G-I) or (J-O) , dorsal views, anterior to the top. (G,J) In WT siblings, the pineal precursors (arrowhead) have converged to form a round pineal anlage. In mutants the pineal precursors (arrowheads) form a domain that is (H,K) elongated or (I,L) divided in two. The pineal anlagen of the (M) mutant and the (N) Zmutant have a round shape that is similar to that of WT fish, while the pineal precursors of the (O) MZmutant are divided in two domains. All images are dorsal views with anterior to the top. Scale bars: 100 μm (A-C, G-I), 70 μm (D-F), 30 μm (J-O)

Nodal signaling is required for closure of the anterior neural tube in zebrafish-5

<p><b>Copyright information:</b></p><p>Taken from "Nodal signaling is required for closure of the anterior neural tube in zebrafish"</p><p>http://www.biomedcentral.com/1471-213X/7/126</p><p>BMC Developmental Biology 2007;7():126-126.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2214732.</p><p></p> live at 95% epiboly (95%), the 4–5 somite stage (4–5 s), the 10 somite stage (10 s), or (G, H) fixed at ~24 hpf. (A-F) Cross sections through the anterior developing neural plate, with presumptive eyes (e) indicated in panel B, the outer boundary of the neural tube indicated by a dotted line in panels B and C, and the midline indicated by the open arrowheads in C. (G, H) Horizontal sections through the midbrain and hindbrain of ~24 hpf embryos, with the midline of the brain (open arrowheads) and the otic vesicles (o) indicated in panel G. (I-J') Embryos were injected at the one cell stage with DNA encoding mGFP, raised to ~24 hpf, and imaged live in high magnification horizontal sections. Open arrowheads indicate elongated cells and the closed arrowheads indicate round cells. All images are confocal optical sections. Scale bars: 40 μm (A-H), 80 μm (I-J')

Nodal signaling is required for closure of the anterior neural tube in zebrafish-6

<p><b>Copyright information:</b></p><p>Taken from "Nodal signaling is required for closure of the anterior neural tube in zebrafish"</p><p>http://www.biomedcentral.com/1471-213X/7/126</p><p>BMC Developmental Biology 2007;7():126-126.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2214732.</p><p></p>ely 24 hpf, and fixed and processed for whole mount in situ hybridization with a probe for . (A) WT and (B, C) some N-cad overexpressing MZembryos have a round shaped pineal morphology indicative of a closed pineal organ. (D, E) Other N-cad overexpressing MZfish have an elongated or divided pineal organ, demonstrating that their NTD has not been corrected. All images are dorsal views with anterior to the top. The background in Panel C has a pink cast because the embryo was too fragile to remove from the yolk. The midline of the brain is apparent in panels A and B, and is indicated by a black line. Scale bar: 40 μm (A-E)

Nodal signaling is required for closure of the anterior neural tube in zebrafish-0

<p><b>Copyright information:</b></p><p>Taken from "Nodal signaling is required for closure of the anterior neural tube in zebrafish"</p><p>http://www.biomedcentral.com/1471-213X/7/126</p><p>BMC Developmental Biology 2007;7():126-126.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2214732.</p><p></p>isense probe for the gene , which is expressed in the pineal precursors (arrowheads). Images are dorsal views of the entire embryo, anterior to the top. (A) At the 2–3 somite stage, pineal precursors are located in two widely spaced lateral domains. (B) By the 5–6 somite stage, these domains have moved towards the dorsal midline of the forebrain. (C) By the 7–8 somite stage, a single, round-shaped pineal anlage has formed. (D-F) At 2 dpf, mutant embryos from the same clutch have a wide range of eye phenotypes. Frontal views of live embryos with dorsal to the top. (D) The eyes of WT embryos and some mutants (not shown) are completely separated from one another. (E) Other mutants have partially fused eyes that form two lenses or (F) a single eye with one lens. (G-L) Embryos at the (G-I) 7–8 somite stage and (J-L) 24 hpf were processed for whole mount in situ hybridization with a probe for the pineal gene (G-I) or (J-O) , dorsal views, anterior to the top. (G,J) In WT siblings, the pineal precursors (arrowhead) have converged to form a round pineal anlage. In mutants the pineal precursors (arrowheads) form a domain that is (H,K) elongated or (I,L) divided in two. The pineal anlagen of the (M) mutant and the (N) Zmutant have a round shape that is similar to that of WT fish, while the pineal precursors of the (O) MZmutant are divided in two domains. All images are dorsal views with anterior to the top. Scale bars: 100 μm (A-C, G-I), 70 μm (D-F), 30 μm (J-O)

Generation and characterization of Kctd15 mutations in zebrafish

<div><p>Potassium channel tetramerization domain containing 15 (Kctd15) was previously found to have a role in early neural crest (NC) patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs) to generate null mutations in zebrafish <i>kctd15a</i> and <i>kctd15</i>b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, <i>kctd15a/b</i> double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa), a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus <i>kctd15</i> mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates.</p></div