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.00402

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 Dorsky, R. I.
Right arrow Articles by Chitnis, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dorsky, R. I.
Right arrow Articles by Chitnis, A.
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?

Two tcf3 genes cooperate to pattern the zebrafish brain

Richard I. Dorsky1,*,{ddagger}, Motoyuki Itoh2,*, Randall T. Moon3,{dagger} and Ajay Chitnis2,{dagger},{ddagger}

1 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84132, USA
2 Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
3 Howard Hughes Medical Institute/Department of Pharmacology and Center for Developmental Biology, University of Washington, Seattle, WA 98195, USA



View larger version (44K):

[in a new window]
  		Fig. 1. tcf3b encodes a protein highly homologous to Hdl and is expressed throughout zebrafish embryogenesis. (A) Clustal alignment of Hdl and Tcf3b amino acid sequences. Dark-gray shading indicates identity and light gray indicates conservative substitutions. Homology is distributed throughout the proteins, but they are most divergent near the C terminus. (B) RT-PCR analysis shows that tcf3b is expressed maternally [0-2 hours post-fertilization (hpf)] and zygotically. max, which is expressed constantly throughout development (Schreiber-Agus et al., 1993Go), was used as a loading control.

 


View larger version (99K):

[in a new window]
  		Fig. 2. hdl and tcf3b show redundant and unique domains of expression during development. (A,B) Shield stage, animal pole view, dorsal towards the right. Expression of hdl is much higher than tcf3b throughout the epiblast at this stage. (C,D) Bud stage, rostral view, dorsal is towards the right. Both genes are expressed in the presumptive forebrain and midbrain and at the ventral midline. (E,F) Bud stage, caudal view, dorsal towards the left. Both genes are expressed at a low level in the notochord, but only hdl is expressed in the tailbud (tb). (G,H) Closer view of head region at six somites. Both genes are expressed throughout the CNS, but tcf3b levels are higher in stripes corresponding to telencephalon and midbrain (mb). (I,J) Six somites, caudal view, dorsal towards the left. Only hdl is expressed in the tailbud and presomitic mesoderm (psm). (K,L) Eighteen somites. Both genes continue to be expressed throughout the brain, with tcf3b showing more specific domains of localization. There is a noticeable gap in tcf3b expression at the midbrain-hindbrain boundary (mhb). sh, shield.

 


View larger version (69K):

[in a new window]
  		Fig. 3. hdl and tcf3b morpholinos (MOs) produce different phenotypes and ectopic expression of tcf3b can rescue the hdl MO phenotype. (A) The hdl and tcf3b MOs can specifically block translation of expression constructs in vitro, in a dose-dependent manner. The hdl MO has no effect on a tcf3b expression construct, and the tcf3b MO has no effect on a hdl construct. (B) Uninjected embryo at 72 hours post-fertilization (hpf). (C) Embryos injected with 1 ng of hdl MO have no eyes or telencephalon, similar to hdl mutant embryos. (D) Embryos injected with 1 ng of tcf3b MO have normal early brain patterning, but overall head size appears smaller (compare with B). (E) When 1 ng of both MOs are injected simultaneously, embryos develop with a much smaller head than with either MO alone (compare with C,D). (F) Rostral view of pax2.1 expression at 90% epiboly in an uninjected embryo, dorsal towards the right. (G) In hdl MO-injected embryos, pax2.1 expression is expanded rostrally. (H) When tcf3b mRNA is co-injected with the hdl MO, pax2.1 expression is restored to the normal domain of two stripes. (I) Normal morphology is observed at 24 hpf after co-injection of tcf3b mRNA and hdl MO. ov, otic vesicle.

 


View larger version (111K):

[in a new window]
  		Fig. 4. Loss of hdl and tcf3b function leads to progressive loss of rostral gene expression domains and concomitant expansion of more caudal domains. All embryos are shown in rostral view with dorsal towards the right. (A1-A3) pax6 expression at 1-3 somites. (B1-B5) pax2.1 and (C1-C5) gbx1 expression at tailbud stage. In hdl mutants, rostral pax6 expression occupies a smaller domain (A2, bar), pax2.1 shows moderate expansion (B2), whereas gbx1 is unchanged (C2). hdl embryos injected with tcf3b MO show a range of phenotypes, suggesting further caudalization of the embryos. Moderately caudalized embryos show complete loss of rostral pax6 (A3), further expansion of pax2.1 (B3) and expansion of gbx1 (C3) expression. In more severely caudalized embryos, pax2.1 expression begins to be lost (B4) as gbx1 expands further (C4). In the most severely caudalized embryos, pax2.1 expression is completely lost (B5) and gbx1 expression shifts to a more rostral domain (C5). (D) Simultaneous examination of pax6 (rostral purple), pax2.1 (red) and gbx1 (caudal purple) expression at the tailbud stage. (E) gbx1 (purple) expression expands rostrally around the pax2.1 (red) expression domain in moderately caudalized embryos.

 


View larger version (109K):

[in a new window]
  		Fig. 5. Wnt8 is required to antagonize Tcf3-mediated repression but not to activate caudal genes. (A-C) Animal pole views, dorsal towards the right. (A) At shield stage, hdl is expressed in the neurectoderm where Wnt signaling is low. (B) By contrast, lef1 is expressed in the ventrolateral mesoderm where Wnt signaling is high. (C) Reduction of Tcf3 function results in expanded lef1 expression. (D,E) Wild-type pax6 expression at one to three somites. Arrowheads in E define the caudal limit of pax6 in the forebrain and rostral limit of pax6 in the hindbrain. (F) Wild-type pax2.1 expression at one to three somites. (G-I) In the absence of hdl function, pax6 is shifted rostrally (arrowheads) and rostral expression is reduced, whereas pax2.1 is expanded rostrally. Changes in the intensity of caudal pax6 expression did not correlate with changes in other caudally expressed genes (see Fig. 4). (J-L) In the absence of both hdl and wnt8 function, pax6 is still reduced and shifted rostrally (arrowheads) and pax2.1 is still expanded. The neural plate is wider in these embryos because of the role of wnt8 in dorsoventral patterning. (M-O) Loss of wnt8 function results in caudal expansion of pax6 (arrowheads) and a caudal shift in pax2.1.

 


View larger version (105K):

[in a new window]
  		Fig. 6. Loss of tcf3b function affects brain morphogenesis. All embryos are shown at 24 hours post-fertilization (hpf), rostral towards the left. (A) Alexa Fluor 594 phalloidin staining reveals forming rhombomere boundaries (arrowheads). (B) In a tcf3b MO-injected embryo, no boundaries are visible and ectopic tissue is present dorsally (arrows). (C) At higher magnification, cell shape changes are apparent at rhombomere boundaries (asterisks) in an uninjected embryo. (D) These features are absent in an injected embryo. (E) wnt1 is expressed at rhombomere boundaries (arrowheads). (F) This expression is disorganized following tcf3b MO injection, and ectopic expression is present medially (arrows). (G) mariposa is normally expressed at ventral rhombomere boundaries (arrowheads). (H) In injected embryos, mariposa expression is uniform and no boundary expression is visible. (I) en2 expression is present throughout the entire midbrain-hindbrain boundary (mhb) when viewed as an optical cross-section. Lateral expression is in mesoderm. (J) After tcf3b MO injection, the mhb fails to close at the dorsal (left) side. (K,L) When viewed dorsally, the rostrocaudal size and position of en2 expression is normal in injected embryos. (M,N) The positions of rhombomeres 3 and 5 are also normal, as marked by krox20 expression. (O,P) isl1 expression indicates that neurogenesis is grossly normal in tcf3b MO-injected embryos. ov, otic vesicle.

 


View larger version (40K):

[in a new window]
  		Fig. 7. Models illustrate how Tcf3 function shapes gene expression in the neurectoderm. (A) By antagonizing Tcf3-mediated repression, a gradient of Wnt/ß-catenin signaling (blue line) transforms early broad expression of Hdl/Tcf3b (unbroken black line) to a rostrocaudal gradient of effective repression (broken black line). Progressive loss of Hdl/Tcf3b (unbroken gray lines) results in changes in the effective repression gradient (broken gray lines). (B) The gradient of Tcf3-mediated repression (broken black line) alters the efficacy of caudalizing factors that are distributed in a gradient (unbroken purple line) to define a gradient of effective caudalizing activity (broken purple line). Discrete windows of effective caudalizing activity regulate the expression of genes that define blue, green, yellow and red compartments. Moderate (C) to severe (D) reduction in Tcf3 function alters the gradient of effective caudalizing activity and expands caudal domains at the cost of rostral domains. (E) A ventrolateral source of Wnts and other caudalizing factors establishes a caudalizing activity gradient with its low end just dorsal to the animal pole. Darker shades of purple represents lower levels of caudalizing activity. (F) A gradient of BMP activity [black (highest) to white (lowest)] is established by the shield (orange) and determines the neurectoderm (broken line). (G,H) In the neurectoderm the gradient of caudalizing activity is interpreted to define discrete neural compartments (blue, green, yellow and red). (I-L) Illustrations of how gene expression domains would be altered with progressively higher effective caudalizing activity. AP, animal pole; V, ventral.

 

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