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

First published online 8 October 2003
doi: 10.1242/dev.00575

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 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 Knight, R. D. Articles by Schilling, T. F. Search for Related Content PubMed PubMed Citation Articles by Knight, R. D. Articles by Schilling, T. F. Social Bookmarking                         What's this?

lockjaw encodes a zebrafish tfap2a required for early neural crest development

¹ Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA
² Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076, Tübingen, Germany

View larger version (55K): [in a new window]   Fig. 2. lowts213 is a mutation in a zebrafish tfap2a. (A) The tfap2a gene is closely linked to the lowts213 locus. Both map to the long arm of linkage group 24 between SSCP markers z23011 and z3399. (B) lowts213 creates a stop codon in the conserved DNA-binding/dimerization domain of tfap2a, resulting in a truncated protein. (C) PCR products of 424 bp were amplified from exon 5 of tfap2a from preparations of individual 3 dpf low and sibling larvae. Digests by BlpI show co-segregation of the restriction site with the low phenotype producing bands of sizes 208 bp and 217 bp. (D) Unilateral rescue of melanophore development at 26 hpf by injection of a PAC containing tfap2a into one blastomere at the two-cell stage (arrows indicate rescued melanocytes). (E) Genomic organization of tfap2a and the locations of morpholino antisense oligonucleotides directed against splice acceptor sites. (F,G) Splicing defects in tfap2a transcripts following injection of splice-directed morpholinos. tfap2a was amplified from pools of 3.1mo (lanes 1-4) and 5.1mo (lanes 5-8) morpholino injected and control uninjected (lanes 9-12) animals using primers tfap2a-3f and tfap2a-3r directed to exons 3 and 7 (lanes 1,2,4-6,9,10) and primers tfap2a-4f and tfap2a-3r directed to exons 4 and 7 (lanes 3,4,7,8,11,12). Uninjected animals (lanes 9-12) showed PCR products of sizes 730 bp and 420 bp, in comparison with morpholino-injected animals in which additional PCR products were observed (black arrows) because of aberrant splicing (F). (G) These produce a phenotype similar to lowts213. Scale bar: 100 µm.

View larger version (78K): [in a new window]   Fig. 1. Pigment, cartilage and aortic arch defects in lowts213 mutant zebrafish. Living wild-type and low mutant embryos. Lateral views (A-C) showing melanocyte reduction at 28 hpf (note normal eye pigmentation in low) and jaw defect at 72 hpf (C). (D-G) Dissected pharyngeal cartilages from 4 dpf wild-type (D,F) and low mutant (E,G) larvae, stained with Alcian Blue and shown in ventral (D,E) and lateral (F,G) view. (E,G) Branchial cartilages have been torn away. (H,I) Camera lucida drawings of mandibular and hyoid cartilages in wild type (F) and low mutant (G). Pharyngeal cartilages and joints (arrows) are absent or reduced in all but the mandibular arch in low mutants, which fuses with the anterior basicranial commissure of the skull. (J,K) Aortic arches in 4 dpf wild-type (J) and low mutant (K) larvae, labeled with fluorescent microspheres and shown in ventrolateral view. (L) Camera lucida drawings of aortic arches in wild-type (left) and defects in low mutants (right). abc, anterior basicranial commissure; cb1-5, ceratobranchials 1-5; ch, ceratohyal; e, eye; hs, hyosymplectic; ih, interhyal; m, Meckel's; op, opercular bone; pc, parachordals; pq, palatoquadrate; t, trabeculae. Scale bars: 100 µm.

View larger version (102K): [in a new window]   Fig. 3. tfap2a expression in early zebrafish embryos. Whole-mount in situ hybridization was performed with an antisense probe to tfap2a. (A) Lateral and (B) animal pole views of expression in the non-neural ectoderm (nn) of an 8 hpf gastrula-stage embryo. Arrows indicate boundary with neural ectoderm. (C) Lateral and (D) dorsal views, anterior towards the left, of expression in the cranial neural crest at 12 hpf (D) and at nine somites (C). Arrowheads indicate premigratory spinal neural crest. (E) Lateral view, 24 hpf; higher magnification is shown in F of segmental expression in the hindbrain and pharyngeal arches. (G-I) Dorsal views of expression in successively more posterior regions of the CNS. (G) Expression in the telencephalon and mesencephalon. (H) Segmental clusters of neurons in each rhombomere. (I) Expression in Rohon-Beard cells in the dorsal spinal cord. (J,K) Lateral views of tfap2a expression in premigratory neural crest in wild type (J) and a low mutant (K). (L) tfap2a expression in midline neural crest and in lateral mesoderm in the tailbud at nine somites (arrow). b, branchial; fb, forebrain; hb, hindbrain; h, hyoid; m, mandibular; mb, midbrain; nc, neural crest; nn, non-neural ectoderm; ot, otic vesicle; r1-7, rhombomeres; rb, Rohon-Beard cell; sc, spinal cord. Scale bars: 100 µm.

View larger version (63K): [in a new window]   Fig. 4. Early neural crest defects in lowts213 mutants. Two-color, whole-mount in situ hybridization was performed at the six-somite (A-F) or 12-somite (G-L) stages, with antisense RNA probes to (A-D) foxd3 and ctn, (E,F) ctn alone, (G,H) sox9b, and (I-L) sna2 and ctn in wild-type (A,C,E,G,I,K) and lowts213 mutant (B,D,F,H,J,L) embryos. Lateral (A,B,F,I,J) and dorsal (C,D,G,H,K,L) views, anterior towards the left. Arrows indicate the otic vesicle. e, eye; mb, midbrain; hb, hindbrain; r3-6, rhombomeres 3-6; sc, spinal cord. Scale bars: 100 µm.

View larger version (97K): [in a new window]   Fig. 5. Defects in pigment precursors, neuronal and glial derivatives of the neural crest. (A-H) Whole-mount in situ hybridization with riboprobes to dct (A,B) and kit (C,D) at 22 hpf (lateral views), as well as gch (E,F) at 25 hpf (dorsal views) and foxd3 (G,H) at 28 hpf (lateral views) in wild-type (A,E,C,G) and lowts213 mutants (B,D,F,H). (I-L) In situ hybridization for isl1 in wild type (I,K) and mutants (J,L) at the six-somite stage (dorsal views). (M-R) Immunostaining with anti-Hu antibody at 72 hpf (lateral views) of wild type (M,O,Q) and mutants (N,P,R) showing cranial (M,N) and spinal (O,P) ganglia (arrows), and enteric neurons (Q,R, arrows). (S,T) Immunostaining with anti-TH antibody of carotid bodies (arrows) at 60 hpf (ventral views) in wild type (S) and mutant (T). e, eye; mn, motoneurons; n, notochord; ot, otic vesicle; rb, Rohon-Beards, V, trigeminal ganglia, X, vagal ganglia. Scale bars: 100 µm.

View larger version (61K): [in a new window]   Fig. 6. Defects in neural crest migration and survival in lowts213 mutants. (A-D) Living 24 hpf embryos labeled by laser activation of DMNB-caged fluorescein (green) 12 hours earlier in the dorsal midbrain and posterior hindbrain of wild type (A) and low mutant (B), or anterior hindbrain of wild type (C) and low mutant (D), and corresponding neural crest (arrows). (E,F) TUNEL labeling of dying cells in six-somite stage zebrafish embryos. (G,H) Co-labeling with TUNEL and in situ hybridization for ctn mRNA at the eight-somite stage (dorsolateral views just posterior to the otic vesicle, ot) showing dying cells within the normal ctn expression domain and in adjacent non-neural ectoderm (arrows). (I) Histogram comparing numbers of apoptotic cells in wild type (blue) and lowts213 mutants (red). Scale bars: 100 µm.

View larger version (77K): [in a new window]   Fig. 7. Defects in pharyngeal arch development in lowts213 mutants. Whole-mount in situ hybridization with riboprobes to dlx2 (A-D) and hoxa2 (E,F) at 28 hpf, as well as gsc (G-J) at 40 hpf. Wild type (A,C,E,G,I); mutant (B,D,F,H,J). Lateral views, anterior towards the left. Arrows in C-F indicate the hyoid arch. Arrows in I and J indicate the dorsal expression domain of gsc, which is absent in lowts213. b, branchial arches, fb, forebrain, h, hyoid arch, hb, hindbrain, m, mandibular arch, mb, midbrain, ot, otic vesicle. Scale bars: 100 µm.

View larger version (71K): [in a new window]   Fig. 8. Transplants suggest that lowts213 functions cell autonomously in neural crest. Prospective hyoid arch neural crest and hindbrain cells at the level of rhombomeres 4 and 5 (r4 and r5) were transplanted from donor embryos labeled with a lineage tracer (red) into unlabeled hosts at 12 hpf (A-C). Lateral views (A'-C') and ventral views (A''-C'') of mosaic larvae at 65 hpf stained for biotinylated tracer (brown). (A-A'') Control transplants between wild-type embryos. (B-B'') Wild-type transplants into low mutants. Arrow indicates rescued hyoid arch outgrowth on the transplanted side. (C,C') Transplants of low mutant crest cells into wild-type hosts. (B''') Rescued muscles of the hyoid arch in low mutants containing transplanted wild-type neural crest. am, adductor mandibulae muscle; P2, pharyngeal arch 2 (hyoid arch). Scale bars: 100 µm.

View larger version (29K): [in a new window]   Fig. 9. low (tfap2a)-dependent and -independent aspects of neural crest development. (A) Zebrafish larva (72 hpf) and the locations of cell types severely disrupted in low mutants [and therefore largely tfap2a dependent (red)] and others that are less effected (blue). (B) Putative roles of tfap2a in gene regulation in the premigratory crest and specification of subpopulations of neural crest derivatives (red).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter