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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02367

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Required, tissue-specific roles for Fgf8 in outflow tract formation and remodeling

¹ Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.
² Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.
³ Institute of Molecular Medicine, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
⁴ Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
⁵ Children's Health Research Center, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.
⁶ Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.

View larger version (87K): [in a new window]   Fig. 1. Differential activity of MesP1Cre, Isl1Cre and Mef2cAHFCre relative to Fgf8 expression domains in cardiac crescent and early pharynx. (A-G) Anti-GFP immunohistochemistry in Fgf8GFP/+;HPRTCre/+ embryos reveals the onset and location of all Fgf8 expression in the cardiac crescent and pharynx. (A-C) Whole-mount preparations show Fgf8GFP is already broadly expressed in the cardiac crescent (cc) by the LHF stage, and in the heart tube (HT) and AHF/SM at 5ss. (D,E) 6ss; transverse sections, anterior to posterior through the heart tube. Cells in the HT (white arrowheads), AHF/SM (yellow arrowheads), foregut (FG), first pouch endoderm (E, red arrowheads) and surface ectoderm (EC, blue arrowheads) are GFP positive. (F,G) 10ss (combined DIC/epifluorescence); Fgf8GFP is present in pharyngeal epithelia, SM and RV/OFT (yellow arrowheads), but not in the left ventricle (LV, white arrowhead). (A1-G1) Fgf8GFP expression in Fgf8GFP/+;MesP1Cre/+ embryos. (A1-C1) Whole-mounts demonstrate Fgf8GFP broadly in the cardiac crescent at LHF and 1ss, and in the HT at 5ss. (D1,E1) 5ss transverse sections; Fgf8GFP is in the HT and SM; endoderm is GFP-negative (gray arrowhead). (F1,G1) 12ss transverse sections; the LV and atrium are now Fgf8GFP negative (white arrowheads). (A2-G2) Fgf8GFP expression in Fgf8GFP/+;Isl1Cre/+ embryos. (A2-C2) Isl1Cre is not active in the cardiac crescent at 0ss (A2), but Fgf8GFP is present at 2ss (B2) and is widely expressed at 5ss (C2). (D2,E2) Pouch endoderm, SM, and ventral HT cells in 5ss embryos are Fgf8GFP positive. (F2,G2) 11ss; transverse sections reveal Fgf8GFP in pharyngeal epithelia, SM and OFT/RV but not in the LV or atrium (A). (A3-G3) Fgf8GFP/+;Mef2cAHFCre embryos. Mef2cAHFCre is not active at 0ss and little GFP is detected in AHF mesoderm until 3ss (B3). Note the lack of expression in any ventral HT cells (D3); expression is restricted to the AHF/SM (E3) and RV/OFT (F3). (H-O2) Comparison of MesP1Cre and Isl1Cre recombination using the Rosa26R-Cre reporter and ß-galactosidase staining. (H-J) Whole mounts MesP1Cre-recombined at 0, 7ss (ventral views) and 34ss (right lateral). (K-K2) 7ss transverse sections of a MesP1Cre/Rosa26R embryo; recombination is evident throughout the head mesoderm (HM), SM and HT, and is restricted to mesoderm. (L-N) Whole mounts of Isl1Cre/Rosa26R embryos. At 1ss (L), few crescent cells are labeled. (M) 6ss, ventral view; black lines depict plane of sections shown in panels O-O2. (N) 24ss, right lateral view; pharyngeal arches and entire RV/OFT are labeled. (O-O2) 6ss transverse sectioned Isl1Cre/Rosa26R embryo; recombination is evident in the foregut (FG), endoderm (E), SM and many HT cells; scattered ventral myocardial cells that are unstained are indicated by the black arrowhead. Rare pharyngeal ectodermal (EC) cells are stained. (P-S) Schematics comparing the timing and location of Fgf8 ablation by different Cre drivers. (P,Q) Whole mounts at LHF-1ss and 2-3ss; Fgf8 is ablated in all myocardial precursors by MesP1Cre (blue); the onset of Isl1Cre is later and in fewer cells (purple); followed by Mef2c-AHFCre only in the AHF at the 3ss (yellow indicates that all three Cre drivers active). Note that the dorsoventral location of these populations is not depicted. (R) Transverse section at 5ss; Isl1Cre is active in pouch endoderm and surface ectoderm (red); all three drivers recombine the SM/AHF and dorsal HT. MesP1Cre ablates Fgf8 in more ventral HT cells, suggesting that Fgf8 is expressed in both heart fields. (S) Transverse section at 10-12ss; all three drivers ablate Fgf8 in SM and AHF-derived RV/OFT (yellow); Fgf8 is no longer expressed in the LV (gray).

View larger version (84K): [in a new window]   Fig. 2. Ablation of Fgf8 in the cardiac crescent disrupts formation of the heart tube and outflow tract. (A-E) Ventral views of the 10-11ss Fgf8;MesP1Cre allelic series; genotypes are listed above. Heart tubes of Fgf8C/-;MesP1Cre/+ conditional mutants are small (red arrowheads). The mildly affected mutant heart (D) is looping, but all segments are small. Although the severely affected mutant (E) has 10 somites, its heart is the size of the 4-5ss control. Note the normal morphology of Fgf8C/+;MesP1Cre/+ (B) and Fgf8C/- (C). (A'-E') Right lateral views of the E9.5 Fgf8;MesP1Cre allelic series. Superimposed black lines are the same length in each panel. The mildly affected mutant (D') has a short, narrow OFT and a small RV (black outline, red arrowhead). (E') The severely affected mutant has a single dilated ventricle, an incompletely looped heart and no OFT (red arrowheads). (F-J) Ventral views of the 7-8ss Fgf8;Isl1Cre allelic series. Fgf8C/+;Isl1Cre/+ (G) is normal, whereas the accruing RV/OFT of the mildly affected Fgf8C/-;Isl1Cre/+ mutant (I) is small (red arrowhead, this phenotype was not seen in >60 controls). (J) Severely affected Fgf8;Isl1Cre heart (red arrowheads). (F'-J') Right lateral views of the Fgf8;Isl1Cre allelic series at E9.5. Black lines in panels F'-H' are the same length and show normal flexion of the conotruncus/OFT. Mutant pharyngeal arches (pa) are hypoplastic (I',J'). The mildly affected mutant (I') has no visible RV and a short OFT lacking normal flexion (black lines); the OFT arises from the LV. Severely affected mutants (J') have incompletely looped, dilated hearts. (K,K') Fgf8;Mef2cAHFCre mutants at E9.5 and 8ss appear normal. (L) Sectioned E10.0 Fgf8C/+;MesP1Cre/+ control. The OFT arises from the RV (black arrows). (M) Sectioned mild E10.0 Fgf8C/-;MesP1Cre/+ mutant heart with a short OFT and small RV. The OFT arises abnormally from the LV (red arrow). Note the enlarged right atrium (ra, red arrowhead) and small RV. OFT cushion cellularity appears normal. (M') Sectioned, severe/dying Fgf8C/-;MesP1Cre/+ mutant; the single ventricle (v), short OFT, and atrium (a) are all dilated. No RV is present. (N) Sectioned E10.5 Fgf8C/+;Isl1Cre/+ control. The OFT arises from the RV and is beginning to septate; the conotruncal cushions are dense with mesenchymal cells (black arrowheads). (O) Sectioned, mild E10.5 Fgf8C/-;Isl1Cre/+ mutant with a RA enlargement, RV hypoplasia, and the OFT arising from the LV. OFT cushions are hypocellular (red arrowheads). (O') Sectioned, severe E10.5 Fgf8C/-;Isl1Cre/+ mutant. There is a dilated single ventricle and atrium. (P) Fgf8C/+;Mef2cAHFCre control. (P') Sectioned, E10.5 Fgf8C/-;Mef2cAHFCre mutants reveal normal OFT cushion cellularity and mild RV hypoplasia. (Q) Dissected heart/great vessels of a newborn Fgf8C/+;MesP1Cre/+ control. Great vessels are normally related (ao, aorta; pa, pulmonary artery; da, ductus arteriosus); white arrow indicates the normal RV alignment and egress via the pulmonary artery. The aorta is posterior and to the right of the pulmonary artery. (R) A newborn Fgf8C/-;MesP1Cre/+ mutant reveals TGA: abnormal OFT alignment/rotation with the aorta anterior, arising aberrantly from the RV (white arrow) and the pulmonary artery arising from the LV. (R') Bicuspid aortic valve (BAV) in a Fgf8C/-;MesP1Cre/+ newborn. (S,S') Aberrant OFT alignment/rotation in an Fgf8C/-;Mef2cAHFCre mutant manifest as DORV with both the aorta (av, aortic valve black arrowhead) and pulmonary artery arising from the RV. (T) Newborn Fgf8C/-;Isl1Cre/+ mutant with Persistent Truncus Arteriosus (PTA) and right aortic arch (white arrow). rcc, right common carotid artery; lcc, left common carotid artery; lsc, left subclavian artery; TA, truncus arteriosus. (T',T'') Sectioned Fgf8C/-;Isl1Cre/+ mutant reveals an abnormally aligned truncal vessel completely overriding the RV; the LV egress is via a large ventricular septal defect (vsd, black arrowhead). tv, truncal valve.

View larger version (86K): [in a new window]   Fig. 3. Loss of Fgf8 signaling disrupts Erm expression and Isl1 production in the anterior heart field. (A-F) Whole-mount (right lateral) 9-10ss embryos after in situ hybridization with an Erm antisense riboprobe. Genotypes are listed above each column; littermate controls processed with mutants are shown. Black arrowheads indicate the domains of normal Erm expression; red arrowheads indicate regions of decreased expression. (A'-F') Upper row sections are anterior through the developing OFT/RV; bottom sections are at the level of the atrium (AT). (B',C') Two Fgf8C/-;MesP1Cre/+ mutants; the mild mutant (B') has relatively normal Erm expression, but the severely affected mutant (C') has decreased Erm in SM, OFT and ventral endoderm. (D',E') Erm expression is comparable in Fgf8C/+;Isl1+/+ and Fgf8C/+;Isl1Cre/+ embryos. (F') Severe Fgf8C/-;Isl1Cre/+ mutant with decreased Erm expression in ventral endoderm, SM and OFT. Note that endodermal expression is more severely affected than in the Fgf8C/-;MesP1Cre/+ mutants and that ectodermal expression remains intact. (G-I) Isl1 mRNA in the 10ss MesP1Cre series; decreased Isl1 expression is apparent in the mutant (red arrowheads). (J-L) Isl1 mRNA in the 7ss Isl1Cre series; Isl1 expression is markedly decreased in Fgf8C/-;Isl1Cre/+ mutants (L, red arrowheads) relative to Fgf8 C/+;Isl1Cre/+ controls (K). (M-P'') Triple fluorescent immunohistochemistry detects Isl1 protein (green), apoptosis (TUNEL, red) and nuclei (Hoechst, blue). (M,M') Sections at the level of the OFT (M) and atrium (M') of a 10ss Fgf8C/+;MesP1Cre/+ control; endoderm, splanchnic and ventral pharyngeal mesoderm, and myocardial cells accumulating to the OFT stain intensely with anti-Isl1 antibody (green arrowheads). (N,N') Same planes of section as above in an Fgf8;MesP1Cre mutant; the OFT is narrow and midline. The intensity of the Isl1 signal and the number of Isl1-positive (green) cells in the OFT and contiguous SM are decreased (red arrowheads). (M'',N'') Another 10ss control and mutant at the level of the OFT; this mutant has no OFT, no Isl1-positive cells in the heart tube (red arrowheads, HT) and excess apoptosis in the Isl1-positive endoderm and adjacent OFT (white arrowheads). (O,O') 8ss Fgf8C/+;Isl1Cre/+ control; at this stage, the OFT is just beginning to accrue and stains with Isl1 (green arrowheads). (P,P') Fgf8C/-;Isl1Cre/+ mutant. Note the excess apoptosis in the Isl1-positive endoderm (white arrowhead). The OFT is abnormally short and few Isl1-positive cells are present in the SM, HT or atrium (red arrowheads). (O'',P'') The alterations in Isl1 protein are reproducible in another 8ss Fgf8C/+;Isl1Cre/+ control and Fgf8C/-;Isl1Cre/+ mutant; the mutant has few Isl1-positive cells in the HT (red arrowheads) and excess apoptosis in endoderm (white arrowhead). (Q-V') Right lateral views of the pharynx/heart of embryos assayed for Isl1 mRNA. Genotypes are listed above, somite stages at the left. The level of Isl1 mRNA (red arrowheads) correlates with the severity of the cardiac/OFT phenotype in mutants.

View larger version (119K): [in a new window]   Fig. 4. Ablation of Fgf8 in crescent mesoderm increases apoptosis and decreases proliferation in foregut endoderm and AHF mesoderm. Transverse cryosectioned Fgf8;MesP1Cre mutants/controls stained with anti-PHH3 (green, mitotic cells) and Hoechst (nuclei), and for apoptosis (TUNEL, red). Sections proceed anterior (top) to posterior (bottom), indicated by the white arrow. (A,B) 0ss; abundant proliferation and minimal apoptosis are detected in control mesoderm (M), endoderm (E) and neuroectoderm (NE) (A, green arrowheads). The number of proliferating cells in the mutant (B) mesoderm appears to be decreased. (C,D) 2ss; increased apoptosis in mutant (D) endoderm (white arrowheads). (E,F) 4ss; increased apoptosis in mutant endoderm (white arrowheads). Many cells are proliferating in the control heart tube mesoderm (HT, white box, green arrowheads) but only a few in the mutant (red arrowheads). (G-J) 9ss; excess apoptosis in the midline endoderm (white arrowheads) and developing OFT of mutants (H,J, red arrows; see also Fig. 3N'',P''). More proliferating cells are detected in the pharyngeal epithelia (green arrowheads; EC, ectoderm; E, endoderm), SM (yellow arrowheads) and contiguous OFT (yellow arrows) of control. Posterior sections in panels I and J are at twice the magnification of those in G and H. pa1, pharyngeal arch 1.

View larger version (105K): [in a new window]   Fig. 5. Loss of Fgf8 specifically disrupts the Isl1/Mef2c pathway in the AHF. In situ hybridized embryos; genotypes listed at the top, riboprobe and somite stage at the left. (A) Mef2c in 10ss embryos; right lateral view, red arrowheads indicate decreased signal in the OFT and AHF/SM of mutants. (A') Dorsal views; the ring of staining around the nascent OFT is markedly less intense in the mutants (red arrowheads). (B,B') Wnt11 at 20-21ss; close up of right side of the heart. Note the undetectable signal in the OFT myocardium of severe mutants (red arrowheads), and decreased levels in the mild variants (B'); expression is intact in Fgf8C/-;Mef2cAHFCre mutants (black arrowhead). (C-C'') Pitx2 at 15ss; right lateral views in C, transverse sections in C',C''. Upper panels are at the level of the OFT, lower panels at level of the atrium/sinus venosus. (D,D') Tbx1 expression in transverse-sectioned 9-10ss embryos appears normal in the endoderm and SM/AHF. (E,E') Fgf10 in transverse-sectioned 8ss embryos. Expression is intact (black arowheads) except in mutant proximal OFT myocardium (red arrowheads).

View larger version (132K): [in a new window]   Fig. 6. Loss of Tbx1 function specifically disrupts the expression of Fgf receptor 1 in the caudal pharyngeal arches. (A-C) Whole-mount in situ hybridized 35ss embryos of the indicated genotypes. Note the Tbx1 dose-dependent reduction in Fgfr1 expression in arches 3-6 (red arrowheads and bracket), and the preserved expression in arches 1 and 2, and the limb bud. This finding was consistent in 6/6 heterozygotes and mutants assayed. (D-E'') Sections reveal decreased Fgfr1 expression in unsegmented endoderm/mesenchyme of the mutant (red arrowheads) compared with the control (black arrowheads).

View larger version (30K): [in a new window]   Fig. 7. Schematics illustrating cardiovascular phenotypes resulting from the differential ablation of Fgf8. (A) Normal Fgf8 expression (green denotes normal Fgf8 expression domains). (B) Ablation of Fgf8 in mesodermal precursors of the heart tube (red) and AHF (blue) at the crescent/early somite stages generates small heart tubes and prevents accrual of the RV/OFT myocardium, usually causing death. (C-E) Compared with wild type (C), loss of mesodermal Fgf8 in surviving Fgf8;MesP1Cre mutants and in Fgf8;Mef2cAHFCre mutants disrupts OFT alignment resulting in TGA and DORV (D). Absence of Fgf8 in the endoderm and mesoderm disrupts septation and alignment, respectively, resulting in PTA with an abnormally aligned truncal vessel (E).