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

First published online May 23, 2006
doi: 10.1242/10.1242/dev.02408

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 Ilagan, R. Articles by Meyers, E. N. Search for Related Content PubMed PubMed Citation Articles by Ilagan, R. Articles by Meyers, E. N. Social Bookmarking                         What's this?

Fgf8 is required for anterior heart field development

¹ Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA.
² Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
³ Department of Anatomy, School of Medicine, University of California at San Francisco, San Francisco, CA 94143, USA.
⁴ Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
⁵ Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX 77030, USA.

View larger version (44K): [in a new window]   Fig. 1. Fgf8 hypomorphs have variable length outflow tracts. (A-D) Fgf8neo/lacZ embryos that undergo relatively normal gastrulation demonstrate OT of variable lengths from mild (B) to moderate and severe (C,D) when compared with control at E9.5 (A). OT, outflow tract; V, ventricle; 1, first pharyngeal arch.

View larger version (130K): [in a new window]   Fig. 2. Comparison of Fgf8 mRNA expression and Fgf8lacZ ß-gal activity relative to AHF development. (A) Fgf8 mRNA, as seen by in situ hybridization, is expressed in the presumptive AHF, but not the primary (1°) heart field (arrow) as early as E7.75. (B,D,F,H,J) Fgf8lacZ expression, as seen by ß-gal activity, is observed in AHF cells from E7.75 through E10.5 in whole mount. (C,C') Expression is later seen in the OT and the contiguous SM at E9.0 in whole-mount (C) and mid-sagittal section (C'). Fgf8 mRNA levels in the OT and SM become reduced at E9.5 (E,E'), and is no longer detectable in AHF cells at E10.5 (G,I). Fgf8lacZ expression at E8.5 in whole-mount (K) and in transverse sections (K1-K3). ß-Gal activity is noted in the SM (black arrowheads) and in the outflow tract (OT). Mid-sagittal sections of Fgf8lacZ embryos reveal ß-gal activity in the PE and SM at E9.0. At E9.5, ß-gal activity is noted in the SM and OT (F'). At E10.0, Fgf8lacZ-positive cells are observed in the pharyngeal CM in whole mount (L) and in coronal section (L'), which are contiguous with the OT. OT, outflow tract; SM, splanchnic mesoderm; CM, core mesoderm; PE, pharyngeal endoderm; LV, left ventricle; SV, sinus venosus.

View larger version (96K): [in a new window]   Fig. 3. Fgf8lacZ expression reflects endogenous Fgf8 expression. ß-Gal activity in Fgf8lacZ/+ embryos at E9.5 (A) is detected in the OT and RV, and areas of normal Fgf8 mRNA expression, such as the brain. ß-Gal activity correlates with the expression of lacZ mRNA seen in whole mount in situ hybridization at E9.5, but very low levels are seen within the OT and RV (B). Fgf8 transcripts are present in the OT at very low levels compared with limb bud controls and HPRT positive controls (C) shown by semi-quantitative RT-PCR analysis at E9.5. These data suggest that Fgf8lacZ expression accurately reflects Fgf8 mRNA expression and that ß-gal activity is more prominent in OT/RV than either lacZ or Fgf8 mRNA. OT, outflow tract; LB, limb bud; NEG, negative control tissue.

View larger version (108K): [in a new window]   Fig. 4. Tissue-specific ablation studies using Nkx2.5Cre and TnT-Cre lines. Fgf8lacZ expression at E8.5 (A,A') overlaps with expression of Nkx2.5Cre in the OT, SM and PE (B,B'). Additionally, Nkx2.5Cre and Fgf8lacZ expression coincides in the CM at E9.5 (D-D'' compared with E-E''). TnT-Cre expression overlaps with Fgf8lacZ only in the OT myocardium (C,C',F-F''). Noted domains of Nkx2.5Cre recombination that coincide with Fgf8lacZ include the OT, SM, CM and lateral PE, but not surface ectoderm [except for first arch (D',D'',E',E'' and data not shown)]. In situ hybridization analysis for Fgf8 was performed at E8.5 (G,H) and E9.5 (I,J) to confirm genetic ablation in these regions with residual expression in the ectoderm. Fgf8 mRNA is markedly reduced in the mutants (H,J) compared with normal controls (G,I). OT, outflow tract; SM, splanchnic mesoderm; PE, pharyngeal endoderm; A, atria; V, ventricles.

View larger version (59K): [in a new window]   Fig. 5. The anterior heart is truncated in Nkx2.5Cre/+; Fgf8flox/- mutants. Analysis of normal (A,C,E,G,I) and mutant (B,D,F,H,J) embryos at E9.5 was performed by whole mount in situ hybridization (A-H) or lacZ stain (I,J) for the genes as indicated. Mhc is expressed in the myocardium. Wnt11 is normally expressed in the OT (C, black arrowhead), but is not expressed in mutants (D, white arrowhead). The boundary of Anf expression in mutants (E,F, black arrowhead) is in close proximity to the aortic sac, indicating virtual absence of OT/RV. Mlc2v-negative myocardium of the proximal OT (G, black bracket) is absent in the mutant (H, white arrowhead). Bmp4lacZ-expressing cells are essentially absent in the OT (J, white arrowhead) and splanchnic mesoderm (J, white bracket) compared with normal controls (I, black brackets). 1, first pharyngeal arch; OT, outflow tract; LV, left ventricle; RV, right ventricle; A, atrium.

View larger version (90K): [in a new window]   Fig. 6. Nkx2.5Cre lineage and Fgf8lacZ-expressing cells are reduced in Nkx2.5Cre/+; Fgf8flox/-; R26R and Nkx2.5Cre/+; Fgf8flox/lacZ mutants, respectively. (A-F) Whole-mount Nkx2.5Cre lineage analysis from E8.5 to E9.5 indicates a drastic reduction of lacZ-positive cells in the mutant class (B,D,F, black arrowheads) when compared with normal controls (A,C,E, black brackets). Histological sections at E9.5 (E',F') reveal a reduction of the Nkx2.5Cre lineage in the pharyngeal endoderm (PE) in addition to the splanchnic mesoderm (SM), both of which are thinned with apparent reduced cell number. Ventral views (G,H) clearly demonstrate the reduction of the Nkx2.5Cre lineage in the CM and SM. Fgf8lacZ-positive cells are also markedly reduced, but present, in SM and OT in the mutant class at E9.5 (J,J') compared with controls (I,I'). Pharyngeal arches are referred to by their respective numbers (1, 2, 3/4); OT, outflow tract; LV, left ventricle; CM, core mesoderm; V, ventricle.

View larger version (100K): [in a new window]   Fig. 7. Cell signaling analysis in Nkx2.5Cre/+; Fgf8flox/- mutants. Immunohistochemistry analysis demonstrates pErk is localized in the SM, but not the PE (A,A'). Nkx2.5Cre elimination of Fgf8 results in loss of pErk from the SM (B,B'). RNA in situ hybridization demonstrates that Pea3 is expressed in the developing arches as well as SM and PE (C,C'). Loss of Fgf8 results in continued arch ectoderm expression (D, black arrowhead) but loss of SM and PE expression (D'). Ptch1lacZ ß-gal activity (E-F') and Shh expression (G,H) demonstrate that the SM and PE are still capable of Ptch1 expression, and that Fgf8 does not regulate this pathway in these tissues. Bmp4 is downregulated within the SM (I,J), suggesting a requirement for FGF8 signaling. OT, outflow tract; SM, splanchnic mesoderm; PE, pharyngeal endoderm; Nt, neural tube/notochord.

View larger version (40K): [in a new window]   Fig. 8. Cell proliferation and survival are reduced in Nkx2.5Cre/+; Fgf8flox/- mutants. Cell proliferation rates at E9.0 were assessed in section by anti-phospho-histone H3 immunostaining (red) in normal (A) and mutant embryos (B). SYTO13 counterstain (green) marks nuclei. Marked reduction in proliferation was noted in the PE and SM of mutants compared with controls (n=3 and 4, respectively). Statistical analysis indicates that decreases in cell proliferation in the PE and SM were significant (P<0.05), while rates in the OT, RV, LV and atria were unaltered (G). LysoTracker Red staining at E9.0 in normal (C) and mutant (D) embryos in confocal optical sections (white signal indicates cell death). Increases in cell death are observed in the SM contiguous with the outflow tract (OT) of mutant embryos. Additionally, increases in cell death are observed in the PE at the level of the OT. LysoTracker Red staining at E9.5 (E,F; red signal indicates cell death) demonstrate that mutant embryos (F) have excess death of NCCs in the pharyngeal arches (1,2). Embryos were counterstained with anti-AP2 (green). OT, outflow tract; SM, splanchnic mesoderm; PE, pharyngeal endoderm; RV, right ventricle; LV, left ventricle; O, otocyst; E, eye.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter