spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00437

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lampert, J. M.
Right arrow Articles by von Lintig, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lampert, J. M.
Right arrow Articles by von Lintig, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Provitamin A conversion to retinal via the ß,ß-carotene-15,15'-oxygenase (bcox) is essential for pattern formation and differentiation during zebrafish embryogenesis

Johanna M. Lampert1, Jochen Holzschuh2, Susanne Hessel1, Wolfgang Driever2, Klaus Vogt1 and Johannes von Lintig1,*

1 Neurobiology and Animal Physiology, Institute of Biology I, University of Freiburg, D-79104 Freiburg, Germany
2 Developmental Biology, Institute of Biology I, University of Freiburg, D-79104 Freiburg, Germany



View larger version (114K):

[in a new window]
  		Fig. 1. (A) Linear alignment of the deduced amino sequence of ß,ß-carotene-15,15'-oxygenases of different vertebrate species. Identity is indicated in black and conserved amino acids are indicated in gray according to the PAM250 matrix. Conserved histidine residues probably involved in binding the co-factor Fe2+ are marked by asterisks. A putative polyene chain oxygenase family motif is underlined. Besides the zebrafish sequence, the following sequences were used: AAG15380 (human), NP067461 (mouse) and AJ271386 (chicken). (B) Color shift of bcox-expressing E. coli-strain caused by the cleavage of ß-carotene to retinal. (C) HPLC profile at 360 nm of an extract derived from a ß-carotene-synthesizing E. coli strain expressing bcox separated on HPLC-system 1. The composition of the individual retinoids is indicated in the figure.

 


View larger version (127K):

[in a new window]
  		Fig. 2. Expression of bcox in zebrafish wild-type and mobm819 embryos and larvae. (A,B,F,H,J-M,O) Lateral views, anterior towards the left; (I) dorsal view, anterior towards the left; (C) optical section of a whole-mount embryo; (G,N) sagittal and (D,E,P,Q) transverse sections. At segmentation stages, bcox is expressed along the AP axis close to the yolk boundary (A, compare with B); representative 14-somite embryos are shown. (C) A view anterior to the trunk at the level of the anterior somites and (D,E) cross-sections show expression in the vegetalmost cell layers adjacent to the yolk, corresponding to mesendoderm and mesoderm. (F) bcox expression in the head mesendoderm and in the ventral part of the optic primordia (see arrow) in a 18-somite embryo. (G) Cross-section through the optic primordium of a 21-somite embryo. (H,I) At 1 dpf, expression is found in the pharyngeal region, in individual cells around the eye, in subcutaneous cells of the head and trunk, as well as in the pectoral fin buds. (J,J') In the zebrafish mutant mobm819 (J') bcox expression is absent in the trunk (compare with J), and (K') highly reduced in the head (compare with K), reflecting the impairments in neural crest development of this mutant (Barrallo et al., 2003Go). (L-N) At 2 dpf, bcox expression is found in the pharyngeal region (N), in the fin buds (L) and in single cells around the eyes (M). (O-Q) In larvae (O), bcox expression is found in the liver (P) and the gut (Q). (R) Western blot analysis of Bcox protein levels in protein extracts of zebrafish embryos. A single band with a molecular mass of 59 kDa was detected. As a control, recombinant murine Bcox (64 kDa) was used. ep, eye primordium; g, gut; hb, hindbrain; li, liver; nc, notochord; ov, otic vesicle; pe, pigment epithelium; pec, pectoral fin bud; ph, pharynx; sc, subcutaneous cells.

 


View larger version (25K):

[in a new window]
  		Fig. 3. Carotenoid and retinoid contents of zebrafish eggs and larval eyes. (A) Retinoid composition of the zebrafish eggs. A HPLC profile at 360 nm of an extract isolated from 180 zebrafish eggs separated on HPLC system 1. (B) Carotenoid composition of zebrafish eggs. A HPLC-profile at 450 nm of an extract isolated from 165 eggs separated on HPLC system 2. (C) Carotenoid and retinoid contents of zebrafish eggs and larval eyes (4 dpf). The values give the means of at least two independent experiments. The individual retinoids and carotenoids were analyzed and quantified on HPLC systems 1 or 2, respectively. Molar amounts were calculated by equilibration of the HPLC systems with defined amounts of authentic reference substances.

 


View larger version (70K):

[in a new window]
  		Fig. 4. Morphological phenotype of bcox morphants. (A-D) Lateral views of 4 dpf bcox morphants raised from eggs injected with 1.5 ng (B), 9 ng (C) and 18 ng (D) bcox-MO compared with a non-treated sibling (A). The bcox-MO treatment provoked malformations, i.e. a curved neck, smaller eyes and an edematically enlarged pericardial region (see arrows) occurred dose dependently in repeated independent experiments. (E,F) The pigmentation of bcox morphants (F) is altered when compared with controls (E). As indicated by arrows, the iridophores are lacking. (G) Western blot analysis of Bcox protein levels in protein extracts derived from 2-day-old bcox- MO-treated embryos and wild-type siblings.

 


View larger version (100K):

[in a new window]
  		Fig. 6. Impairments of the development of the craniofacial skeleton in bcox morphants. (A-D) Alcian Blue staining of the cartilage of the craniofacial skeleton of bcox morphants (B,D) when compared with controls (A,C). The Meckel's cartilage and palatoquadrates are deformed, the ceratohyales project posteriorly, and the ceratobranchials are reduced or absent (see asterisks). (E) Co-injection of bcox-MO with bcox mRNA rescued the morphant phenotype (arrows indicates the restored cartilage). (F,G) The pectoral fins of bcox morphants are shortened and project laterally. (H,I) ap2{alpha} expression in the pharyngeal region at 24 hpf (lateral view) is reduced in cranial neural crest in bcox morphants when compared with controls (see arrows). At 31 hpf (J,K dorsal view), dlx2 expression is reduced in the posterior arch primordia in bcox morphants (K, see arrows). At 2 dpf (L,M, lateral view), dlx2 expression is altered in anterior arch primordia, whereas it is reduced in the posterior arch primordia in bcox morphants (M) when compared with controls (L; see arrow in M). At 3 dpf (N,O, lateral view), dlx2 expression persisted in the pharyngeal region in bcox morphants (O, see arrow) whereas it is absent in controls (N). (P,Q, lateral view) Application of exogenous RA (10–7 M) results in a downregulation of dlx2 expression in pharyngeal region of 3 dpf bcox morphants (Q, see arrow). ch, ceratohyal; m, Meckel's cartilage; nc, neurocranium; pec, pectoral fins; pq, palatoquadrate. In all pictures, anterior is towards the left.

 


View larger version (99K):

[in a new window]
  		Fig. 5. Analyses of hindbrain development of bcox morphants. The expression patterns of krx20 (A,B) efna4 (C,D) and val (E,F) reveal no obvious alterations in the identity of rhombomeres 1 to 5 in bcox morphants when compared with controls (14 hpf/24 hpf, dorsal views). Whole-mount in situ hybridization for combinations of krx20 and myod (G,H, 14 hpf, dorsal view), as well as fgf8 and val (I,J, 20 hpf, lateral view) reveal no alterations in the distance between r5 and the first somite or between the midbrain hindbrain border and r5 (the bar and the curve give the distances found in wild-type embryos, respectively). (K,L, 22 hpf, lateral view) hoxb4a expression is reduced in bcox morphants (L) when compared with controls (L). (M,N, 22 hpf, lateral view) Treatment with 10–9 M RA restores hoxb4a expression in bcox morphants (N). (O,P, 24 hpf, lateral view; Q,R, 31 hpf, dorsal view) In bcox morphants (P,R), ap2{alpha} expression is reduced along the whole rostrocaudal expression domain in hindbrain and spinal cord, and is absent at the level of the otic vesicle. (S, dorsal, T, lateral) Citral treatment of wild-type embryos provoked similar changes in ap2{alpha} expression as found in bcox morphants. ov, otic vesicle; r, rhombomeres; teg, tegmentum.

 


View larger version (75K):

[in a new window]
  		Fig. 7. Impairments of the eyes of bcox morphants. (A,D) pax6.1 expression at 30 hpf in wild-type embryos, (B,E) bcox morphants and (C,F) citral-treated wild-type embryos (lateral and dorsal views). As indicated by arrows, pax6.1 expression is particularly reduced in the ventral retina of bcox morphants and citral-treated wild-type embryos. (G,H) The pax2.1 expression domain is enlarged in the optic stalk of bcox morphants (H) when compared with controls (G). (I,J) shh expression is not altered in bcox morphants (J) when compared with controls (I). (K,L) Cross-sections through the eyes of bcox morphants (L) and control siblings (K) at the same magnification. (K',L') The insets show a higher magnification of the outer layers of the retina. Note the shortened and less stratified outer segments of the photoreceptors in bcox morphants (L'). (M) Retinoid composition of the eyes of 4 dpf larvae of wild type (upper trace) when compared with bcox morphants (lower trace). The figure shows the profiles at 360 nm of extracts isolated from 80 eyes per trace and separated on HPLC system 1.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003