QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00544

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tian, J. Articles by Sampath, K. Search for Related Content PubMed PubMed Citation Articles by Tian, J. Articles by Sampath, K.

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

Jing Tian^1,2, Caleb Yam², Gayathri Balasundaram¹, Hui Wang^1,*, Aniket Gore^1,2 and Karuna Sampath^1,2,

¹ Laboratory of Fish Embryology, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604
² Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543
^* Present address: Department of Biology, University of Virginia, Charlottesville, VA, USA

View larger version (60K): [in a new window]   Fig. 1. Variable fusion of the eyes (A-E) and ventral curvature (F-J) in protruding mouth stage (6 days post fertilization at 22°C) cycsg1 mutant embryos maintained at 22°C. Class I (A,F) represents the mildest phenotype similar to wild-type embryos, and class V (E,J) represents the most severe phenotypes, similar to null mutations in cyc. At 28.5°C, only class V phenotypes are observed.

View larger version (98K): [in a new window]   Fig. 2. Identification of the molecular lesion in cycsg1. (A) Nucleotide sequence electropherogram showing an A to T transversion at position 853 of the coding sequence of cyc. (B) The mutation introduces a new site for the restriction enzyme PvuII, seen in digests of DNA amplified from individual cycsg1 mutant embryos, when compared with wild-type embryos. (C) Schematic representation of the leader, pro and mature ligand domains of Cyc, with the cleavage site and Arg-Stop change indicated (black arrowhead). Expression of shh in control embryos (D) compared with expanded (E) or multiple (F,G) domains in embryos injected with cycsg1 mutant RNA (E-G) and incubated at 22°C. Arrowhead in G indicates an additional axis in the posterior. (D-G) Dorsal views. (H-L) Expression of wild-type Cyc+FLAG protein (I) compared with Cycsg1FLAG mutant protein (K,L) in animal cap explants incubated at 22°C (K) or 28.5°C (L), and control explants (H). Cos-7 cells transfected with pCS2cyc+FLAG (J) or pCS2cycsg1FLAG (M) show localization of Cyc+FLAG protein (J) but not of Cycsg1FLAG(M). The weak nuclear staining in pCS2cycsg1FLAG transfected cells was detected in untransfected controls as well (not shown). White arrowheads indicate cells expressing high levels of protein. Scale bars: in L, 20 µm for H,I,K,L; in M, 20 µm for J,M.

View larger version (78K): [in a new window]   Fig. 3. Expression of cyc and gsc transcripts in cycsg1 mutant embryos. (A-F) Dorsal views; (G-J) anterior views. At 70% epiboly (A-C) as well as 90% epiboly (D-F), cyc transcripts in cycsg1 mutants (B,E) at 22°C are similar to wild-type embryos (A,D). At 28.5°C, cyc transcript levels are reduced in cycsg1 mutant embryos at 70% epiboly (C), and not detected by 90% epiboly (F). Compared with wild-type embryos (G) or cycsg1 mutant embryos at 28.5°C (H), gsc expression is variably reduced in cycsg1 mutant embryos at 22°C (I,J).

View larger version (72K): [in a new window]   Fig. 4. Floor-plate cells are present in cycsg1 mutant embryos at 22°C but not at 28°C. (A-D) Dorsal views; (E-H) lateral views with anterior towards the left. Expression of the early marker of floor-plate cells, twhh, in 100% epiboly wild type (A) and cycsg1 mutants at 22°C (B,C) compared with lack of expression in cycsg1 mutants at 28.5°C (D). At prim-5 stage, cycsg1 mutants have patchy to complete shh expression in the floor plate at 22°C (E-G) and lack of floor plate shh expression at 28.5°C (H). (E) Gaps in the expression of shh in the ventral brain and spinal cord (red arrowheads). In E-H, dotted boxes mark the area displayed in the inset with patchy shh expression in the floor plate of the trunk and a normal notochord underneath (white arrowheads). (F) Gaps in shh expression in the hindbrain and rostral spinal cord (red arrowheads) but fairly complete floor plate in the trunk (inset). (G) Nearly complete ventral brain and rostral spinal cord floor plate, and patchy shh expression in the trunk (inset, red arrowheads). (H) At 28°C, similar to cyc null alleles, shh expression is seen in the notochord (white arrowhead), but not in the overlying neural tube except for a few cells in the dorsal midbrain (yellow arrowhead).

View larger version (95K): [in a new window]   Fig. 5. Primary and secondary motoneurones in cycsg1 mutant embryos. (A-D) Cross-sections at the level of the trunk. (E-G) Anterior views. (H-J) Lateral views of trunk. (A-D) Expression of isl2 (purple, asterisks) marks the position of primary motoneurones in the ventral neural tube. Compare shh (red) expression in the floor plate (black arrowheads) and notochord (white arrowheads) in wild-type embryos (A) with that in cycsg1 mutants at 22°C (B-D). Retinal ganglion cell axons (E-G) and secondary motoneurones (H-J) in wild type (E,H) or cycsg1 mutants at 22°C (F,I) or 28°C (G,J). Black arrows indicate axonal projections from retinal ganglion cells and secondary motoneurones.

View larger version (31K): [in a new window]   Fig. 6. Temperature shift experiments indicate that the floor plate is induced during gastrulation. (A) In shift-up experiments, embryos transferred from 22°C to 28.5°C after 75% epiboly show patches of shh-expressing (shh+) floor-plate cells (C), which increased significantly if the shift was performed at 80% epiboly and later stages (D). (B) In experiments where embryos were shifted down from 28°C to 22°C, embryos shifted at 50% epiboly and 60% epiboly had complete expression of shh in the floor plate (E). The number of embryos with shh+ floor-plate cells, and the extent of rescue, decreases if the shift-down is performed later during gastrulation (F). Lateral views are shown.

View larger version (44K): [in a new window]   Fig. 7. Temperature pulse experiments reveal the precise time window of floor-plate induction. (B-E) twhh expression, dorsal view, (F-I) shh, lateral view. (A) While incubation at 22°C between 70 and 80% epiboly was sufficient to induce floor plate fates in >90% mutant embryos, the extent of rescue was groups of cells distributed throughout the neuraxis (D,H). Maximal rescue was observed in the embryos kept at 22°C between 60 and 90% epiboly (B,F). In the converse experiment, embryos pulsed at the restrictive temperature (28.5°C) showed very few (E,I) floor plate cells in the 70-80% interval or no floor plate cells (C,G) if pulsed at 28.5°C between 60 and 90% epiboly.

View larger version (84K): [in a new window]   Fig. 8. Induction of the early floor-plate gene, twhh, requires high levels of Cyc/Nodal signalling. (A-L) Dorsal views at 50-60% epiboly. (A-D) flh; (E-H) gsc; (I-L) twhh. (A,E,I) Control embryos showing expression of all marker genes in the shield. Overexpression of 0.05 pg of cyc+ RNA results in expansion of the flh domain (B), but not of gsc (F) or twhh (J). Injections of higher doses of cyc+ RNA result in expansion of the gsc (G,H) and twhh (K,L) domains as well.

View larger version (21K): [in a new window]   Fig. 9. Specification of floor-plate cells is dependent on high levels of Cyclops signalling. Embryos from matings of cycsg1 (A), cycsg1 with cycm294 (B) and cycsg1;sqtcz35 (C), and of cycsg1;sqtcz35 with cycm294;sqtcz35 (D) heterozygous fish were maintained at 22°C or 28°C, and analysed for shh expression to determine the extent of floor plate, and hgg1 expression for the prechordal plate mesendoderm at prim-5 stage. (A) Mutants homozygous for cycsg1 showed the expected proportion of embryos with shh-floor plate at 28°C, whereas at 22°C, all mutant embryos were shh+ for the floor plate through the entire length of the ventral neuraxis. (B) Embryos harbouring one null copy of cyc in combination with one copy of cycsg1 showed a small proportion of mutant embryos with no shh expression in the floor plate domain, even at 22°C (4/79). However, the proportion of shh- embryos at 22°C was always less than that seen in the siblings from the same mating maintained at 28°C. In addition, 18 mutant embryos at 22°C showed patches of shh+ floor-plate cells. (C) In embryos from matings of cycsg1;sqtcz35 transheterozygotes, at 22°C no shh-hgg+ embryos (genotype cyc) were detected, compared with 17% in the siblings kept at 28°C. In addition, the proportion of embryos that were lacking both shh and hgg1 expression is significantly less at 22°C than that seen in embryos from the same clutches kept at 28°C. (D) In comparison with C, shh-hgg1+ embryos were seen in matings of cycsg1;sqtcz35 with cycm294;sqtcz35 heterozygotes, even at 22°C. In addition, the proportion of shh–hgg1- embryos was comparable with that seen in siblings from the same mating at 28°C.