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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00850

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Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development

¹ Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
² Childrens Health Research Center, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
³ Program in Molecular Biology, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
⁴ Program in Neuroscience, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
⁵ Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
⁶ Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84112, USA

View larger version (22K): [in a new window]   Fig. 1. Nonhypomorphic, conditional reporter alleles of Fgf8 used for domain-specific ablation experiments. (A) Different cassettes were inserted into the 3' UTR of Fgf8 to generate the Fgf8APN and Fgf8GFPN alleles. An in-frame splice acceptor and the alkaline phosphatase (AP) or green fluorescent protein (GFP) reporter genes (green boxes) were positioned downstream of the 3' loxP site (red arrowheads) and an frt-flanked neor gene (green bars flanking labeled arrow) in a SpeI site located in the 3'UTR of Fgf8. These alleles are hypomorphic because the presence of neor. Flp-mediated recombination (purple arrow) of frt sites (green bars) deletes the neor gene and generates nonhypomorphic conditional reporter alleles, Fgf8AP and Fgf8GFP. Fgf8AP and Fgf8GFP alleles are inactivated with respect to production of functional Fgf8 message when Cre (large red arrow) recombines the loxP sites (red arrowheads) to delete exon 5 (Moon and Capecchi, 2000). Recombination results in expression of the AP or GFP reporter gene under control of Fgf8 regulatory sequences (Fgf8APR, Fgf8GFPR), allowing detection of functionally relevant recombination of Fgf8 (i.e. inactivation of Fgf8 in cells in which it is expressed).

View larger version (78K): [in a new window]   Fig. 2. The Ap2-IRESCre driver ablates FGF8 in the developing pharyngeal arch (PA) ectoderm. (A) A 12 kb genomic fragment containing exons 6, 7, the 3' UTR and polyadenylation signal from the Ap2 locus was used to generate the targeting vector for homologous recombination in ES cells. (B) The targeted allele contains the IRESCre cassette (see Materials and methods), positioned 198 bp 3' of the translation stop in an engineered AscI site. (C) AP2-IRESCre was tested for Cre activity by crossing to the Rosa26lacZ reporter strain. 12, 21 and 35 somite stage embryos, with the genotype AP2IRESCre/+; Rosa26lacZ/+, were assayed for ß-galactosidase activity (blue staining). Somite stages (ss) are labeled in the lower right corner of each panel and PAs are numbered. Blue staining, and both yellow and red arrowheads denote regions of Cre activity in the ectoderm of the PAs as they develop; Cre activity in the caudal ectoderm that will form PAs 3-6 is highlighted by the large red arrowhead in the 12 and 21 ss embryos. (D) Functionally relevant recombination of the Fgf8AP conditional reporter allele in the developing PA ectoderm by the AP2-IRESCre driver. A whole-mount, 21 ss Fgf8AP/+;AP2-IRESCre/+ embryo is shown in the left panel after assaying for alkaline phosphatase activity. The black line indicates the plane of the coronal section shown in the right panel. The ectoderm of developing PA 3, and that of PAs 1 and 2, are stained violet because of Fgf8APR activity. Although AP2-IRESCre is expressed in neural crest, the ectomesenchyme of the PAs is not stained because Fgf8 is not expressed in these cells. (E) AP2-IRESCre ablates Fgf8 function throughout its expression domains in the PA ectoderm. Expression of Fgf8GFPR after recombination with the AP2-IRESCre (left panel) versus universal `deleter' Cre driver (right panel) (Schwenk et al., 1995), was assessed by whole-mount anti-GFP immunohistochemistry of 22 ss, stage-matched embryos (ss in lower right corner). The domains of Fgf8 inactivation resulting from the AP2-IRESCre driver are the same ectodermal domains detected with the universal `deleter' Cre. Fgf8GFPR is expressed in caudal ectoderm that will form PAs 3-6. (F,G) Coronal sections through PAs 1 and 2, and the developing third arch region of a 20 ss embryo. Fgf8GFP/+;AP2IRESCre/+ embryo (G) reveals Fgf8GFPR expression throughout the ectoderm of the developing third arch and cleft.

View larger version (97K): [in a new window]   Fig. 3. hoxa3-IRESCre ablates Fgf8 function in the third and fourth PA endoderm and ectoderm. (A) 11 kb of murine hoxa3 genomic sequence extending from a 5' Sau3AI site to an EcoRI site 3' of the stop codon in exon 2 was used to create the targeting vector. (B) The targeted allele contains the IRESCre cassette (red box) inserted in an ApaI site four bases 3' of the stop codon. Function of the hoxa3-IRESCre allele assayed with the Rosa26lacZ reporter. (C) Whole-mount and sectioned preparations of Rosa26lacZ/+; hoxa3IRESCre/+ embryos stained for ß-galactosidase activity (blue staining) at the 10, 16, 20, 23 and 26-28 ss reveal the caudal-to-rostral progression of hoxa3-IRESCre activity and anterior limit at the rhombomere 4/5 boundary and the anterior border of PA3. The relatively dorsal plane of the coronal sections through the PAs is demonstrated by the black line labeled d in whole-mount panel 20. These results demonstrate hoxa3-IRESCre activity in all three tissue layers of developing PAs 3-6. PAs are numbered; ss are noted in lower right corner of each panel. 2p, PA2 pouch; en, endoderm. (D,E) Functionally relevant recombination of Fgf8GFP in the developing PA epithelia by hoxa3-IRESCre occurs throughout the Fgf8 PA3-6 epithelial expression domains. (D) Coronal sections through the PAs of different stage Fgf8GFP/+; hoxa3IRESCre/+ embryos were assayed for GFP using fluorescent immunohistochemistry. Somite stages (ss) are labeled in the lower right corner of each panel followed by a letter indicating relative plane of each section within the arch of interest; v, ventral; d, dorsal (plane illustrated in Fig. 3C, whole-mount panel 20, black lines labeled v or d). PAs are numbered; 3p, PA3 pouch, etc. Fgf8GFPR expression is initially detected throughout the epithelia of developing PA3 and then is lost from the rostral endoderm (yellow arrowheads, sections 24v, d and 27v, d). Because the Rosa26lacZ reporter studies in Fig. 3C indicate that hoxa3-IRESCre is active throughout the endoderm of PA3 from the 20 ss, this loss of Fgf8GFPR expression in rostral endoderm reflects a change in the Fgf8 expression domain, not failure of hoxa3-IRESCre expression in these cells. (E) GFP expression was assayed in coronal sections of Fgf8GFP/+; deleterCre embryos. Stage-matched embryos to those shown in D were assayed for GFP. Note that the expression of GFP from the globally recombined Fgf8GFPR allele entirely recapitulates that seen in the PAs of Fgf8GFP/+; hoxa3IRESCre/+ embryos, including the loss of expression at later stages in the rostral endoderm of PA3 (yellow arrowheads, sections 24v,d and 27v,d). These studies confirm that hoxa3-IRESCre ablates Fgf8 function throughout its PA epithelial expression domains from at least 20 ss. Labels are as noted for D.

View larger version (107K): [in a new window]   Fig. 4. Ablation of FGF8 in the PA ectoderm with AP2-IRESCre reveals a unique and required role for FGF8 during PA vascular development, separate from its role in glandular and outflow tract development. (A-D) Gross morphology of perinatal control and mutant embryos. (A) Fgf8AP/N newborn control; note that in the absence of any source of Cre, embryogenesis occurs normally with only one functional, nonhypomorphic allele of Fgf8. (B) Fgf8 newborn hypomorphic mutant (Fgf8APN/N); the decrease in Fgf8 mRNA produced by the Fgf8APN allele cannot support normal development resulting in neonatal death, growth delay, edema, cyanosis, craniofacial malformations (red arrowhead) and cardiovascular defects. (C) Fgf8/AP2-IRESCre E18.5 mutant; note absence of the lower jaw (red arrowhead) and bulging eyes, showing that complete ablation of FGF8 in PA1 results in a much more severe craniofacial defect than FGF8 deficiency in the hypomorph (red arrowhead). (D) Fgf8/hoxa3-IRESCre newborn has normal craniofacial development because Cre is not expressed anterior to PA3. (E-I) Thoracic dissections of control and Fgf8/AP2-IRESCre mutant newborns. (E) Wild-type specimen with normal ascending aorta and left aortic arch (Ao, arrowhead), left ductus arteriosus (DA, arrowhead), right common carotid (rcc), left common carotid (lcc), right subclavian artery (rsa), left subclavian artery (lsa), trachea (tr); the right brachiocephalic artery (rbc) branches to form the rsa and rcc. (F) Normal bilobed thymus (th) in wild-type animal. (G) Removal of the thymus (see H) from an Fgf8/AP2-IRESCre mutant reveals interrupted aortic arch type B (IAAB, no transverse aortic arch, black arrowhead), a left DA and an abnormal isolated lsa. (H) The same mutant shown in G has a normal thymus. AP2-IRESCre-mediated ablation of FGF8 separates vascular from outflow tract and pharyngeal gland defects that result from global Fgf8 deficiency. (I) Fgf8/AP2-IRESCre mutant after removal of the thymus; in this case there is a right aortic arch (Ao, arrowhead) and a right DA (arrowhead). (J-L) Transverse sections of a wild-type animal showing the normal progression (slides shown from rostral to caudal) of the head and neck vessels arising from the transverse left aortic arch. (J,K) Normal junction of rbc and lcc to lsc to form the Laa. (L) Normal ascending Ao and junction of DA to descending aorta (dA). (M) Normal right and left coronary arteries (rca, lca) arising from the left and right cusps of the aortic valve. (N-S) Fgf8/AP2-IRESCre mutants have multiple vascular anomalies caused by abnormal formation of the fourth PAAs. (N) Single coronary artery with abnormal origin of lca from right cusp of aortic valve; the rca branches off this vessel in a more caudal location. (O) Circumflex right aa (circ raa) joining lsa after a retroesophageal route; the DA also joins the lsa to form a left descending Ao (not shown). (P,Q) IAAB: the ascending aorta gives rise to the junction of the rbc and lcc (see also E). The transverse aortic arch (P, arrowhead) and descending Ao (Q, arrow) are absent, resulting in an isolated lsa that only connects with the DA (Q) to form the left descending aorta (not shown); this lesion is lethal. (R,S) A different Fgf8/AP2-IRESCre mutant with right aortic arch: the lcc joins the rbc to form the transverse region of the right-sided arch (aa). A right DA, right descending aorta (dA) and an aberrant lsa are also seen in this mutant (S).

View larger version (99K): [in a new window]   Fig. 5. Pharyngeal arch artery defects in Fgf8 domain-specific mutants at E10.5. Stage-matched embryos are shown photographed at the same magnification. (A,B) Ventrolateral views of the right (A) and left (B) sides of an ink injected E10.5 control embryo. Remodeling toward a left dominant system is already evident. Pharyngeal arch arteries (PAAs) are numbered, the dorsal aorta and aortic sac are labeled (DoA, AS respectively). (C,D) An Fgf8/AP2-IRESCre mutant with bilateral aplasia of the fourth and sixth PAAs. The third PAA is abnormally enlarged and provides blood flow from the heart to the DoA (see also Fig. 7, rows K, L). There are sprouts from the DoA visible on the left side (red arrowheads). (E-H) Fgf8/AP2-IRESCre mutants with bilateral hypoplasia of the entire PAA system (E,F), or aplasia of the fourth PAAs (G,H); the red arrowhead highlights a sprout from the AS on the left side.

View larger version (135K): [in a new window]   Fig. 6. Ablation of FGF8 in the PA endoderm and ectoderm by hoxa3-IRESCre reveals that endodermal domain-specific FGF8 activity is required for thymic, parathyroid and aortic valve formation. (A) Sections through neck of E18.5 control. Note the large parathyroid gland (pth) embedded in posterior-lateral aspect of thyroid (tyr). e, esophagus; tr, trachea. (B-D) Parathyroid ectopy and/or hypoplasia in Fgf8/hoxa3-IRESCre mutants. c, clavicle. (E) Wild-type control with normal bi-lobed thymus. (F,G) Thymic hypoplasia and migration defects in Fgf8/Hoxa3-IRESCre mutants. (F) Hypoplastic left thymus in association with a right aortic arch (raa). (G) Monolobed ectopic gland. (H) Cross-section of normal aortic valve in control animal, anterior is towards the left; note three cusps: right (R), left (L), posterior (P). (I-K) Bicuspid aortic valves in Fgf8/Hoxa3-IRESCre mutants. (L) Cross-section through the right ventricular outflow tract of a control embryo showing the relationship of the aorta (Ao), pulmonary valve (PV) and right ventricle (RV). (M-O) Sections through an animal with Tetralogy of Fallot (TOF). (M) Dysplastic PV and severe subvalvar and infundibular stenosis. (N) Cross-section of the left ventricular outflow tract (LVOT), right ventricle (RV) and intact ventricular septum in a wild type animal; there is continuity between the aortic (Ao) and mitral (mv) valves. Note thickness of right ventricular free wall and ventricular septum (yellow bidirectional arrows); the heart is fixed in late systole (mitral valve is closed). (O) The mutant displays overriding aorta (both the right and left ventricular outflow tracts empty through the aortic valve), right ventricular hyperplasia with marked increase in thickness of the right ventricular wall and ventricular septum (yellow bidirectional arrows, heart in late systole), and a large membranous ventricular septal defect (red arrowhead).

View larger version (100K): [in a new window]   Fig. 7. TUNEL analysis of whole-mount and sectioned preparations specimens reveals similar patterns of abnormal neural crest apoptosis in Fgf8 hypopmorphs, Fgf8/AP2-IRESCre and Fgf8/HoxA3-IRESCre mutants. Both experiments were repeated three times with comparable results. The whole-mount experiment was performed with all embryos in the same tube; the sectioned specimens were all processed simultaneously. PAs are numbered; o, otocyst. (A-H) Right and left views of 25-26 ss stage-matched embryos. (A,E) A wild-type embryo has minimal apoptosis in the region of PAs 3-6 (white ovals, white arrowhead). The white line indicates plane of section for fluorescent immunohistochemical analysis shown in I-L. (B,F) An Fgf8 hypomorph replicates our previous finding of abnormal domains of apoptosis in PAs 3-6 (yellow arrowheads) (Frank et al., 2002). (C,G) Fgf8/AP2-IRESCre and (D,H) Fgf8/hoxa3-IRESCre mutants have increased NC apoptosis in the same regions noted in the hypomorph (yellow arrowheads). (I-L) Fluorescent immunohistochemistry was performed cryosectioned 25 ss, stage-matched control versus hypomorphic and domain-specific mutants using a combination of anti-AP2/FITC (green fluorescence) to detect neural crest cells (NC) and TUNEL/Texas red (red fluorescence). Embryos were sectioned transversely in parallel with the third PA (see white line in A). Each row shows a representative section from a single embryo, proceeding from anterior to posterior through the postotic region (from the third PAA to the developing fourth and sixth arch region). Sections were carefully matched to represent the same region of each embryo in each column, taking into consideration the profoundly perturbed anatomy and severe pharyngeal hypoplasia of Fgf8 hypmorphic and Fgf8/AP2-IRESCre mutants. White asterisks indicate the third PAA in parallel (not present in all sections), yellow arrowheads indicate regions of abnormal apoptosis and dying NC. The dorsal aorta is labeled (ao). (I) A control embryo has minimal apoptosis in NC migrating into the third PA or region of the developing fourth and sixth PAs. (J) An Fgf8 hypomorph has large domains of abnormal apoptosis. Double-labeled NC are migrating from rhombomere 6 into the lateral third arch. Note that the pharynx is poorly segmented and that the PAs and third PAA are hypoplastic. (K) Fgf8/AP2-IRESCre and (L) Fgf8/hoxa3-IRESCre mutants have NC apoptosis in the same domains as the hypomorph. In these examples, the third PAA is pathologically enlarged (see also Fig. 5C,D).

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