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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

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 Maroon, H.
Right arrow Articles by Mason, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maroon, H.
Right arrow Articles by Mason, I.

Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle

Habib Maroon1, Jennifer Walshe1, Radma Mahmood1,*, Paul Kiefer2, Clive Dickson2 and Ivor Mason1,{dagger}

1 MRC Centre for Developmental Neurobiology, New Hunt’s House, King’s College London, Guy’s Campus, London SE1 9RT, UK
2 Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
* Present address: Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Bronx, NY 10641, USA



View larger version (84K):

[in a new window]
  		Fig. 1. fgf3 expression in relation to otic placode and otocyst development. (A-H,J-L) Detection of fgf3 mRNA in the developing hindbrain (arrows) of the zebrafish embryo. (A) 90% epiboly. (B) Bud (10 hours). (C) 5 somites (11.75 hours). (D) Flatmount of a 5 somite (11.75 hours) embryo; anterior towards the left. (E) 14 somite (16 hours). (F) 20 somites (19 hours). (G,H) Hybridisation to fgf3 (blue) and krox20 (red) in 4 somite (G) and 14 somite (H) embryos. (I) Detection of hoxb1 (blue) and krox20 (red) in a 12 somite embryo. (J,K) fgf3 (blue) and dlx3 (orange) expression in 3 somite embryos; (K) flatmount. (L) Flatmount showing fgf3 (blue) and pax2.1 (orange) in a 5 somite embryo. (M) Flatmounted 10 somite embryo showing fgf3 (orange) and pax8 (blue). (N) Dorsal view of an embryo at 90% epiboly to show onset of fgf8 expression (arrowhead) in the presumptive r4 territory.

 


View larger version (35K):

[in a new window]
  		Fig. 2. Inhibition of Fgf3 and Fgf8 using morpholinos. (A) Immunoblot detection of Fgf3 in extracts of zebrafish embryos probed with an anti-Fgf3 antibody. Lane 1: zebrafish Fgf3 produced in and purified from a mammalian cell line. Lane 2: Recognition of zebrafish Fgf3 in an extract of four 5 somite embryos. The other crossreacting bands were detected variably from blot to blot and were not sensitive to knockdown by Fgf3mo. (B) Reduction in Fgf3 protein in immunoblots of individual Fgf3mo-injected embryos. Lanes 1-4: detection of Fgf3 in protein extracts of individual 5 somite embryos. Lane 1: uninjected embryo. Lane 2: embryo injected with control Fgf3 morpholino. Lanes 3 and 4: embryos injected with Fgf3mo. (C,D) Fgf8mo phenocopies homozygous ace zebrafish at 26 hours of development. (C) Embryo injected with control Fgf8 morpholino. (D) Embryo injected with Fgf8mo lacks cerebellar tissue. Te, telencephalon; Cb, cerebellum.

 


View larger version (66K):

[in a new window]
  		Fig. 3. Size reduction and loss of otic vesicles in fish injected with Fgf3 and Fgf8 morpholinos. (A-E) 26 hour (prim 8) embryos photographed at the same magnification using Nomarski optics. Where otic vesicles are present (arrows), their maximum diameter (anteroposterior axis) is measured and indicated in red. (A) Embryo injected with control Fgf3 morpholino. (B) Embryo injected with Fgf3mo. (C) Embryo injected with Fgf8mo. (D,E) Embryos co-injected with Fgf3mo and Fgf8mo showing further reduced size (D) or absence of an otocyst (E).

 


View larger version (105K):

[in a new window]
  		Fig. 4. Detection of apoptotic and dividing cells in embryos injected with Fgf3 and Fgf8 morpholinos. Apoptotic (A-L) and dividing cells (M-T) detected in lateral views of zebrafish embryos at 14 somites photographed at low and high magnification (A-H) and in dorsal views of the hindbrain and otic territory of embryos at 8 somites (I-T). (A,B,I) Embryos injected with control Fgf3 morpholino. (C,D,K) Embryos injected with Fgf3 morpholino. (E,F,J) Embryos injected with Fgf8 morpholino. (G,H,L) Embryos injected with both Fgf3 and Fgf8 morpholinos; note large number of apoptotic cells in the dorsal midline of the neural tube in 14 somite embryos. (M-T) Low magnification (M,O,Q,S) and high magnification (N,P,R,T) views of dividing cells in the hindbrains and adjacent otic territory of embryos injected with both control morpholinos together (M,N), Fgf3 morpholino (O,P), Fgf8 morpholino (Q,R) and Fgf3mo together with Fgf8mo (S,T).

 


View larger version (91K):

[in a new window]
  		Fig. 5. Loss of early otic placode markers in embryos injected with Fgf3 and Fgf8 morpholinos. Embryos were hybridised for dlx3 (A-H) and pax2.1 (I-P) after incubation to 2-5 somite (A-D and I-L) or 18-21 somite (E-H and M-P) stages after injection with morpholinos. (A,E,I,M) Embryos injected with control Fgf3 morpholino are indistinguishable from uninjected embryos. (B,F,J,N) Embryos injected with Fgf3mo show reduction in level and extent of dlx3 (B,F) and pax2.1 (J,N) expression. (C,G,K,O) Embryos injected with Fgf8mo show reduction in level and extent of dlx3 (C,G) and pax2.1 (K,O) expression. (D,H,L,P) Embryos injected with Fgf8mo and Fgf3mo together show loss of dlx3 (D,H) and pax2.1 (L,P) expression. Arrows indicate position of dlx3- and pax2.1-expressing cells as appropriate.

 


View larger version (95K):

[in a new window]
  		Fig. 6. Loss of pax2.1 expression in embryos treated with an inhibitor of Fgfr signalling. Embryos were treated with either DMSO (A-C,G-I) or SU5402 (D-F,J-L) for 5 hours between 30% epiboly and tailbud (A,D,G,J), 60% epiboly and 2-3 somite (B,E,H,K), or 5-somite and 16-somite stages (C,F,I,L). Embryos were either fixed immediately and hybridised for erm transcripts (A-F) or allowed to develop until 16 somites and then hybridised for pax2.1 (G-L). (A-F) erm transcripts are reduced or abrogated in the otic region and hindbrain (brackets in A and D, arrows in B,C,E,F). (G-L) pax2.1 transcripts (arrows) are absent in embryos exposed to inhibitor at early stages (G,H,J,K) but not at later stages (I,L).

 


View larger version (53K):

[in a new window]
  		Fig. 7. Induction of pax8 in presumptive otic ectoderm is independent of Fgf signals. (A-D) pax8 transcripts are detected in control embryos (A) and embryos injected with Fgf8 morpholino (B), Fgf3 morpholino (C), and Fgf3 and Fgf8 morpholinos together (D), and allowed to develop to 2 somites. (E-H) Treatment with SU5402 does not abolish pax8 expression. (E,F) Embryos treated between 30% epiboly and tailbud. (E) Control. (F) SU5402 treated. (G,H) Embryos treated between 60% epiboly and 2-3 somites. (G) Control. (H) SU5402 treated. Arrows indicate pax8 expression in presumptive otic territory.

 




© The Company of Biologists Ltd 2002