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

First published online 5 January 2006
doi: 10.1242/dev.02215

This Article Summary Full Text Full Text (PDF) Supplementary Material 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 Pietsch, J. Articles by Shepherd, I. T. Search for Related Content PubMed PubMed Citation Articles by Pietsch, J. Articles by Shepherd, I. T. Social Bookmarking                         What's this?

lessen encodes a zebrafish trap100 required for enteric nervous system development

¹ Department of Biology, Emory University, Rollins Research Center, 1510 Clifton Road, Atlanta GA 30322, USA.
² Department of Developmental Biology, Stanford University School of Medicine, Stanford CA 94305, USA.
³ Department of Biological Structure, University of Washington, Box 357420, Seattle WA 98195, USA.

View larger version (52K): [in a new window]   Fig. 1. lsn-mutant phenotype. (A,B) Lateral views of live larvae at 96 hpf, showing the abnormal development of the jaw, eye and heart in lsn (B) when compared with wild type (A), and lack of swim bladder in lsn. Arrow in A indicates the swim bladder. (C,D) Lateral views of 96 hpf embryos stained with anti-Hu antibody shows normal DRG development in lsn but a reduced number of enteric neurons that are absent from the distal end of the gut tube. Stars indicate the end of the gut tube; arrows indicate DRGs; arrowheads indicate enteric neurons. (E,F) Lateral views of the gut tube of 96 hpf embryos stained with anti-Hu antibody showing reduced number of neurons in lsn.

View larger version (142K): [in a new window]   Fig. 2. lsn mutation affects both craniofacial and thymus development. (A-D) Alcian Blue staining showing pharyngeal cartilages in 120 hpf wild-type (A,C) and lsn mutant (B,D) larvae shown in lateral (A,B) and ventral (C,D) views. In lsn, the development of the pharyngeal cartilages is abnormal. Notably the ceratobranchials 4 and 5 are absent in lsn. cb 1-5, ceratobranchial cartilages 1-5; ch, ceratohyal cartilage; hs, hyosymplectic cartilage; m, Meckel's cartilage; pq, palatoquadrate cartilage. (E,F) rag-1 expression at 96 hpf in wild-type (E) and lsn mutant (F) larvae shows that lsn embryos have a significantly reduced thymic primordia (arrows).

View larger version (29K): [in a new window]   Fig. 3. Positional cloning of lsn. (A) lsnw24 maps on to linkage group 12 between markers z9891 and z4373. No recombinants were found between lsnw24 and a SSCP marker in the 3' UTR of an EST (Fa05b04) that encodes for a zebrafish trap100. We isolated a full-length cDNA corresponding to trap100 from homozygous-lsn mutant and wild-type embryos and 12 independent clones were completely sequenced in both directions. A T to A (encoding Y to stop) mutation was identified in trap100 cDNAs derived from homozygous-lsn mutants. (B) A schematic illustrating the overall structure and percentage similarity between human, mouse and zebrafish Trap100, and the predicted truncated Trap100 in lsn mutants. (C) Sequence tracing of one of the 12 independent clones derived from genomic PCR of genomic DNA from mutant and wild-type embryos showing TAT (Y) to TAA (stop) mutation. (D) A bar graph showing rescue of the lsn homozygous mutant enteric phenotype by injection of wild-type trap100 mRNA. Embryos derived from an incross of heterozygote lsn fish were injected with 50 pg of trap100 mRNA, fixed and stained with anti-Hu antibody at 96 hpf and genotyped. Control represents genotyped uninjected embryos from the same cross. Total number of enteric neurons in a 10-somite segment of the intestine were counted. Numbers represent the mean number of enteric neurons in this 10-somite segment±s.e.m. for 15 embryos of each genotype for each condition. *Significantly different from control (Student's t-test P<0.001).

View larger version (93K): [in a new window]   Fig. 4. Effect of trap100 antisense morpholino oligonucleotide injection on jaw, heart and enteric neuron development. (A,B) Lateral views of the heads of 96 hpf trap100 morphant (A) and lsn mutant (B) embryos. Arrowheads indicate the recessed jaw; arrows indicate cardiac oedema. (C,D) Lateral views of the intestines of 96 hpf embryos stained with anti-Hu antibody. Arrowheads indicate the end of the intestine. The lack of melanophores in morphant the embryo (A) is not due the effect of the trap100 morpholino. Embryos injected with the trap100 morpholino were obtained from an incross of nacre homozygous fish (Lister et al., 1999) and lack melanophores because of a mutation in mitfa.

View larger version (110K): [in a new window]   Fig. 5. Developmental expression pattern of trap100. (A-F) Whole-mount in situ hybridized embryos hybridized with a trap100 antisense probe at the indicated developmental stages. (A-D,F) Lateral views; (E) Dorsal view of 24 hpf embryo. Asterisks in D-F indicates the otic vesicle. Arrows in E indicate fin buds. Arrowhead in F indicates increased expression of trap100 in the posterior mesencephalon. Arrows in F indicate intestinal mesendodermal expression of trap100. (G) A transverse section taken through 48 hpf embryo at the level of somite 4 showing expression through the intestinal mesendoderm. Arrows indicate intestinal mesendodermal expression of trap100. (H) A transverse section taken trough the gut tube at 60 hpf after double in situ hybridization with a trap100 fluorescein antisense probe (red) and a phox2b digoxigenin antisense probe (purple) showing intestinal epithelia expression of trap100. (I) Same section as in H showing trap100 expression using fluorescence in the intestinal epithelia cells. Arrows and asterisks in H,I indicate intestinal mesendodermal expression of trap100and phox2b-positive ENS precursors, respectively. (J,K) Whole-mount in situ hybridized embryos hybridized with a trap100 antisense probe at 72 hpf. (J) Lateral view; (K) dorsal view. Arrowheads in J indicate pharyngeal arch mesendodermal expression. Arrowhead in K indicates ventral diencephalon cells expressing neurons expressing trap100. In all whole mounts (A-F,J,K), anterior is towards the left; (F,K,J) yolk has been removed.

View larger version (100K): [in a new window]   Fig. 6. trap100 is required for normal intestinal development but is not required for endoderm-intestine transition. (A,B) Cross-sections of 120 hpf wild-type (A) and lsn mutant (B) embryos stained with Toluidine Blue. (C,D) trypsin expression at 96 hpf in whole-mount in situ hybridized wild-type (C) and lsn mutant (D) embryos. (E,F) ifabp expression at 96 hpf in whole-mount in situ hybridized wild-type (E) and lsn mutant (F) embryos. p, pancreas; *, liver.

View larger version (126K): [in a new window]   Fig. 7. lsn mutation causes a failure of enteric precursors to populate the entire length of the intestine but does not perturb the initial migration of vagal neural crest to the anterior gut. (A,C,E,G) Wild-type and (B,D,F,H) lsn mutants embryos. Anterior is towards the left. (A,B) Ventral view of the vagal region of 36 hpf embryos hybridized with riboprobes for crestin. (C,D) Ventral view of the vagal region to somite 10 of 48 hpf embryos hybridized with riboprobes for phox2b, showing a failure of phox2b-expressing cells to populate the entire length of the intestine. Arrows indicate the migrating enteric precursors. The yolk has been removed from the embryos (A-D). (E-H) Lateral views of the intestine, 72 hpf wild-type (E,G) and lsn mutant (F,H) embryos hybridized with riboprobes for phox2b (E,F) and nos1 (previously nnos) (G,H).

View larger version (58K): [in a new window]   Fig. 8. lsn mutants have reduced ENS precursor proliferation. (A-D) Confocal images of dissected intestines from 48 hpf wild-type (A,B) and lsn (C,D) embryos immunocytochemically stained with anti-Phox2b antibody (red) and anti-phosphohistoneH3 antibody (green). (A,C) Anti-Phox2b immunoreactivity in wild type (A) and lsn (C), showing the most posterior point along the gut tube that Phox2b-positive cells can be identified. (B,D) Merged images of the intestines in A and C showing anti-Phox2b and anti-phosphohistone H3 immunoreactivity (arrows). Asterisk in A indicates the end of the gut lumen, which is not opened up to the distal end of the gut tube at this developmental age. Arrows in B,D indicate double-labelled cells. (E) Bar graph showing the mean number of Phox2b immunoreactive cells (red) and double-labelled Phox2b/phosphohistone H3 positive cells (green) present along the entire length of the intestine of 48 hpf wild-type and homozygous mutant lsn embryos. Embryos derived from an incross of heterozygote lsn fish were fixed and stained with anti-Phox2b and anti-phosphohistone H3 antibodies at 96 hpf and genotyped. Numbers represent the mean number of immunopositive cells±s.e.m. for 10 embryos of each genotype. The difference between them was statistically significant (Student's t-test, *P<0.001).

View larger version (70K): [in a new window]   Fig. 9. Transplants suggest that lsnw24 functions cell autonomously in the endoderm and endoderm is required for ENS development. (A-D) Transplantation of wild-type endodermal cells partially rescues posterior arch cartilage formation in lsn mutants. (A) At blastula stage, fluorescein-dextran labelled wild-type cells expressing Tar* were transplanted into host and allowed to develop. The lateral view of the head region at 72 hpf shows grafted cells (green) have differentiated into pharyngeal endoderm derivatives. (B-D) Ventral views of 5 dpf larvae stained with Alcian Blue to show cartilages. (B) Wild-type control. All lower jaw cartilages are clearly identifiable. (C) lsn mutant larvae transplanted with wild-type cells showing rescue of posterior ceratobranchial (3-5) in the vicinity of transplanted Tar*-expressing wild type cells (black staining) in the pharyngeal arch endoderm (arrowheads). (D) lsn mutant that lacks posterior ceratobranchials. (E-H) Casanova morphant embryos have no intestinal endoderm and no migrating ENS precursors. Dorsal views of 48 hpf control embryos (E,F) and cas morphant embryos (G,H) hybridized with riboprobes for phox2b (E,G) or foxa3 (F,H) showing a complete lack of phox2b-expressing cells in the region of the intestine of morphant embryos (G) that also completely lack intestinal endoderm (H). Arrowheads in E indicate migrating ENS precursors. m, Meckel's cartilage of the mandibular arch; ch, ceratohyal cartilage; 1-5 ceratobranchial cartilages; G, intestine; L, liver, P, pancreas.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter