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Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse

Radwan Abu-Issa1, Graham Smyth2, Ida Smoak3, Ken-ichi Yamamura4 and Erik N. Meyers1,2,*

1 Department of Pediatrics, Neonatal Perinatal Research Institute, Duke University Medical Center, Durham, NC 27710, USA
2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
3 Department of Molecular Biomedical Sciences, North Carolina State University Raleigh, NC 27606, USA
4 Institute of Molecular Embryology and Genetics, 4-24-1 Kuhonji, Kumamoto, 862-0976, Japan



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  		Fig. 1. (A) Normal anatomy with the aorta (Ao) arising to the left and dorsal to the pulmonary artery (Pa). (B) Fgf8 mutants had frequent transposition with the Ao arising to the right and adjacent to the Pa (black arrowhead), and also an abnormal subclavian artery pattern (white arrowhead). (C) Less frequently there was persistent truncus arteriosus (TA). Note left branch pulmonary artery (LPa) arising from Ao to supply lung (Lu) (white arrowhead in C). In wild type (D), the Ao arises from the left ventricle (LV), while in Fgf8 mutant (E) the Ao arises from the right ventricle (RV). In another mutant (F), a single outflow tract arising from both the RV and LV over-rides a ventricular septal defect (VSD). Fgf8 mutants had atrial septal defects (white arrowhead H) or common atrium (CA) (I). In addition, although normally the right and left ventricles are separated from the right atrium (RA) and left atrium (LA) by the tricuspid (TV) and mitral valves (MV), respectively (G,H), several mutants had a single atrioventricular valve (I, black arrowheads). In addition to these abnormalities, ink injections/inspection of near-term embryos demonstrated a normal sized aortic arch in wild type (white arrowhead in J) although Fgf8 mutants often had hypoplastic (white arrowhead in K) or completely absent (black arrow in L) aortic arch, which is derived from the fourth arch artery. BC, braciocephalic; LCC, left common carotid; LS, left subclavian.

 


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  		Fig. 2. Left side views of whole-mount transgenic Tie2-lacZ (A-C,G-I) (see Materials and Methods) and ink injections (D-F,J-L) demonstrate abnormal arch artery patterning in Fgf8 mutant embryos. At E9.5, (A-F) endothelial cells of the first through third arch arteries are normally well developed in wild type embryos (A), while in Fgf8 mutants the arch arteries were often hypoplastic (arrowheads in B). In addition, the second arch was often proximally interrupted (arrowhead, C). These findings were essentially confirmed by ink injection (compare D with E,F). At E10.5 (G-L), the third, fourth and sixth arch arteries are well formed in wild type (G), while in Fgf8 mutants they were often hypoplastic (H) or completely missing, as the second and fourth are in I (note embryo in I was also ink injected). Similarly, ink injections demonstrate hypoplastic (arrowhead in K) or absent (L) fourth arch artery in Fgf8 mutants when compared with control (J).

 


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  		Fig. 3. Gross morphology of E10.5 embryos demonstrate the first and second arch to be much smaller and fused in Fgf8 mutants (arrows in B, compare with A). Confocal optical sectioning through the arches revealed the absence of pharyngeal clefts 1 and 2 (compare D with C and arrowhead in E with F), while the upper pharyngeal pouches were roughly intact (compare pouches 1 and 2 in D and C). The caudal pharyngeal arches were often absent as well (compare 3 and 4 in E with F). Pax1, which is expressed in the pharyngeal pouch endoderm at E9.5 (G,H) and E10.5 (I,J), is abnormal in Fgf8 mutants (H,J) when compared with controls. Note gap in arch 2 (arrow, H) and loss of expression (arrow, J).

 


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  		Fig. 4. E18.5 skull bone preparations (A-F) of wild-type (A,C,E,G,I) and Fgf8 mutants (B,D,F,H,J) demonstrate abnormal development of the palate (P), palatine (PL), alisphenoid (A) and zygomatic arch (Za). The mandibles of Fgf8 mutants were extremely hypoplastic, sparing only the most distal structures (compare C with D). Defects in the styloid process (Sp), tympanic rings (Tr), stapes, incus (S+I), and malleus (M) are observed as well in mutants (F) when compared with wild type (E). Fgf8 mutants also had abnormal or absent external ears (compare arrow in G with H). Immunohistochemistry for the 2H3 antibody demonstrated that cranial nerves 5, 7, 9 and 10 were fairly well formed in wild type (I), with reduction in Fgf8 mutants (J) of the distal tips (compare arrowheads in I to J), and narrowing of the facial branch of cranial nerve 5 (arrow, J). In addition, nerve 10 had fewer ramifications proximally (compare black arrowhead in I with that in J).

 


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  		Fig. 5. mRNA in situ hybridization for two neural crest markers CrabP1 and Ap2{alpha} revealed a similar NCCs stream between wild type and Fgf8 mutants for second and third PA (arrows in A,B) and first and second PA (arrows in C,D). Lineage trace of NCCs using the transgenic lines P0-Cre and R26R (E,F) (see Materials and Methods) demonstrated decreased NCCs within the PAs. High-powered examination of the outflow tract (red brackets in E,F) also demonstrated that decreased NCCs were entering the outflow tract of Fgf8 mutants (arrowheads, H) when compared with controls (arrowheads, G), despite NCCs having progressed as far into the outflow tract (arrows in G,H). G and H are at the same magnification and embryos had the same number of somites (±2).

 


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  		Fig. 6. Proliferation within the developing arches appeared grossly the same between wild type (A) and Fgf8 mutants (B,C). Examination of cell death between E8.0-E9.5 revealed a striking pattern of increased cell death in Fgf8 mutants. In E8.0-8.5 Fgf8 mutants (E), there was increased death along the flank near the cardiac inflow (arrowheads, E,H) as well as along the dorsal axis (stars, E,H) and anterior neural ridge (E, arrow) when compared with control embryos (D,G). At E9.5, there was increased cell death within the caudal arches (arrowheads, K) and cells entering the arches (N) of Fgf8 mutants when compared with controls (J,M). At E9.5 the pattern of cell death was similar to that of lineage traced NCCs in wild-type embryos using P0-Cre and the Cre reporter R26R. (compare M, N with O). Strikingly, the mRNA expression of Fgf8 in wild-type embryos closely matched the areas of cell death (compare F,I,L with E,H,K, respectively). ot, otocyst.

 


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  		Fig. 7. TUNEL analysis in E10.5 PO-Cre::R26R::Fgf8neo/– mutant demonstrates NCCs that are dying. Frontal section through first PA demonstrates ß-Gal-positive cells (A), TUNEL-positive cells (B) and overlap (C). Blow up of boxed area in C clearly demonstrates double labeled (lacZ and TUNEL positive) cells (D). Nt, neural tube; E, eye; BA1, pharyngeal arch 1.

 


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  		Fig. 8. mRNA in situ hybridization at E9.5 demonstrates loss of Tbx1 expression in the core of the first and second arch in mutant (B) when compared with control (arrows A). In addition there was no expression in the area of the third arch artery (arrowheads, compare A with B). Expression of dHAND was maintained or expanded in Fgf8 mutants (D,F) compared with wild type (C,E). Frontal view of first arch reveals possible expansion of dHAND in Fgf8 mutants (G,H). Expression of Dlx2, (I,J) Msx1 (K,L) and Gli1 (M,N) were reduced or absent in the arches (arrows), particularly in PA2 with very distal sparing in PA1.

 

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© The Company of Biologists Ltd 2002