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First published online 27 July 2005
doi: 10.1242/dev.01945

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Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development

Alan O. Perantoni¹, Olga Timofeeva^1,*, Florence Naillat^3,*, Charmaine Richman², Sangeeta Pajni-Underwood², Catherine Wilson², Seppo Vainio³, Lee F. Dove¹ and Mark Lewandoski^2,

¹ Laboratory of Comparative Carcinogenesis, National Cancer Institute, NCI-Frederick, Frederick, MD 21702, USA
² Cancer and Developmental Biology Laboratory, National Cancer Institute, NCI-Frederick, Frederick, MD 21702, USA
³ Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland

View larger version (79K): [in a new window]   Fig. 1. T-Cre-mediated activation of the R26R reporter allele. (A) Schematic representation of the T-Cre transgene. (B) Cre-mediated recombination of the R26R reporter deletes the neoR fragment thereby restoring ß-galactosidase activity (ß-Gal). (C-L) Whole-mount images (C-F,I) or sections (G,H,J-L) of T-Cre; R26R embryos stained for ß-Gal. (C) Lateral view of an E7.5 embryo. Horizontal bar indicates the extraembryonic/embryonic border. (D) Ventral view of an E8.0 embryo, demonstrating reduced staining in the node region. (E) Dorsal view of an E8.5 embryo. (F) Lateral view of an E9.0 embryo. Dotted lines indicate transverse sections shown in panels G and H. (I) Lateral view of an E10.5 embryo. Dotted lines indicate sections in panels J and K. (J) Horizontal section through an E10.5 head. (K) Transverse section through an E10.5 embryo, demonstrating ß-Gal staining in the ectoderm and AER. (L) Section through an E14.5 kidney. 4th, fourth ventricle of neural tube; aer, apical ectodermal ridge; al, allantois; ß-ac. polyA, DNA fragment containing the ß-actin polyA sequences; cm, cephalic mesenchyme; fb, forebrain; gt, gut; hb, hindbrain; ht, heart; ln, lens; mb, midbrain; mv, mesencephalic vesicle; nc, notochord; nd, node; nt, neural tube; pro, promoter; ps, primitive streak; se, surface ectoderm; so, somites.

View larger version (73K): [in a new window]   Fig. 2. T-Cre-mediated recombination results in Fgf8 inactivation specifically in mesodermal lineages. (A) Genetic cross generating T-Cre; Fgf8Flox/2,3 mutants. (B) Cre-mediated recombination deletes exons 2 and 3, generating the Fgf82,3 null allele. The probe indicated was used in ISH of embryos in panels C-G and hybridizes only to mRNA generated from the unrecombined Fgf8Flox allele. (C-G) Lateral views of whole-mount ISH at ages indicated. Mutant embryos are on the right of, or above, the control. Fgf8 expression is minimally affected in E6.5 mutants (C), but is greatly diminished in E7.5 mutants (D) and absent in E8.0 mutants (E). (F,G) Fgf8 is expressed in neural epithelium and surface ectoderm structures at E8.5 (F) and E9.5 (G), but is absent in somites (asterisks), tailbud and nephrogenic cord (G). aer, apical ectodermal ridge; anr, anterior neural ridge; cp, commissural plate; hp, heart progenitors; hf, headfold; mhb, mid/hindbrain junction; nm, nephrogenic mesenchyme (cord); ps, primitive streak; tb, tail bud. Horizontal bars indicate the extraembryonic/embryonic border.

View larger version (105K): [in a new window]   Fig. 3. Fgf8 expression in control and T-Cre; Fgf8Flox/2,3 kidneys. (A) Urogenital tissues of control and mutant female neonates showing abnormally small kidneys. (B) Whole-mount ISH of Fgf8 in a control 11.5 kidney (left, lateral view; right, dorsal view), demonstrating expression adjacent to the ureteric bud. The ureteric bud and kidney are outlined in white and black dashed lines, respectively. (C) Whole-mount ISH using a Fgf8 probe specific for the Cre-deleted sequences (see Fig. 2B) of the E12.0 urogenital region (ventral view) in control (left) and mutant (right). Brackets indicate kidneys. (D) ISH performed on sectioned E14.5 control kidney. (E) Enlargement of the box in D illustrates Fgf8 expression in renal vesicles and newly formed tubules. (F) Whole-mount ISH using a Fgf8 probe specific for Cre-deleted sequences (see Fig. 2B), demonstrating expression in control (left) and mutant (right) E14.5 kidneys. (G) Whole-mount ISH using an Fgf8 probe including sequences outside of the Cre-deleted region (see Fig. 2B), demonstrating continued Fgf8 expression of Fgf8 in both control (left) and mutant (right) E14.5 kidneys. ad, adrenal gland; bl, bladder; cb, comma-shaped body; gl, glomerulus; ki, kidney; mm, metanephric mesenchyme; ov, ovary; rv, renal vesicle; ub, ureteric bud; ur, ureter; ut, uterus.

View larger version (116K): [in a new window]   Fig. 4. Metanephric development in control (A,C,E,G) and T-Cre; Fgf8Flox/2,3 (B,D,F,H) progeny stained with Hematoxylin and Eosin. (A,B) At E12.5, control (A) and mutant (B) kidneys show similar patterns of ureteric bud (UB) branching and induction of metanephric mesenchyme, with condensation and renal vesicle formation (arrowheads). (C,D) At E14.5, mutant kidneys (D) show renal vesicles, which do not progress, unlike controls (C) in which S-shaped bodies (s) and glomeruli (g) are evident. This pattern persists at E16.5 (E,F), and, by E18.5 (G,H), only radii of UB branches remain in mutants (H). Scale bars: 100 µm.

View larger version (143K): [in a new window]   Fig. 5. Aberrant cell death occurs in T-Cre; Fgf8Flox/2,3 kidneys. Confocal imaging of metanephroi from control (A,C,F) or T-Cre; Fgf8Flox/2,3 (B,D,E,G,H) embryos at E12.5 (A,B), E14.5 (C,D,E), E16.5 (F,G,H) stained with the acidotrophic fluorochrome Lysotracker Red. E and H are enlargements of the boxed regions in D and G, respectively. D shows a preferential loss of mesenchymal cells from the cortical nephrogenic zone (also arrows in D and G). H shows a loss of tubular epithelial cells (arrowheads) in mutant tissues.

View larger version (133K): [in a new window]   Fig. 6. Nephrogenic progenitors are preferentially lost in T-Cre; Fgf8Flox/2,3 kidneys. (A,B,E,F) E14.5 kidneys; (C,D,G,H) E16.5 kidneys. (A-D) Sustained expression of the stromal marker Foxd1/BF2 in sections from whole-mount ISH of control (A,C) and T-Cre; Fgf8Flox/2,3 (B,D) metanephroi. (E-H) Depletion of the cap cell marker Cited1 demonstrated by immunohistochemistry of control (E,G; arrowheads indicate cap formation) and T-Cre; Fgf8Flox /2,3 (F,H) metanephroi.

View larger version (142K): [in a new window]   Fig. 7. Substantial loss of multiple metanephric markers occurs in T-Cre; Fgf8Flox/2,3 kidneys between E14.5 and E16.5. Expression patterns for tissue-specific markers of metanephric differentiation in control and mutant E14.5 and E16.5 metanephroi by thin-section ISH. Arrowheads in the Wt1 panels indicate podocyte expression domains; arrows in the Pax2 panels indicate renal vesicles. Scale bar: 20 µm.

View larger version (118K): [in a new window]   Fig. 8. Prior to phenotypic changes, a loss of Wnt4 (A) and Lim1 (B,C) expression occurs in MM in mutant E12.5 metanephroi. Arrows and arrowheads indicate MM and UB expression domains, respectively.

View larger version (71K): [in a new window]   Fig. 9. The Wnt4–/– nephrogenic phenotype. (A,C,E,G) Control; (B,D,F,H) Wnt4–/–. Metanephroi from E18.5 control (A) and Wnt4–/– (B) embryos. Wnt4–/– metanephroi develop few nephrons (a nephron-like structure is shown in B, inset). (C-H) Wnt4–/– metanephroi also develop few foci of Fgf8 expression (C-E), and express no Lim1 in induced MM (H, compare with G). Arrows and arrowheads indicate MM and UB expression domains, respectively. Scale bar in H: 250 µm.

View larger version (104K): [in a new window]   Fig. 10. A Wnt and Fgf8 signal are required for tubulogenesis rescue in mutant MM. (A,C,E) Embryonic spinal cord (SC) induces tubule formation in metanephric mesenchyme (MM) from control embryos (A), but not from T-Cre; Fgf8Flox/2,3 mutant embryos (C) unless Fgf8-soaked beads are added (E). (B,D,F) Sections of cultured tissue shown on the right.