Figure 1.
Representative bfb phenotypes at developmental age E11.5 (A), E13.5 (B), E15.5 (C) and E17.5 (D).
Blood-filled blisters are evident across the eye and the distal hindlimbs (asterisks), although clear in the rear appendages at E13.5 and often distorting the digits in older embryos (arrowhead in D).
Figure 2.
The bfb mouse harbours a mutation in Fras1.
(A) Chromatogram of Fras1 gene sequence identifying the c.10762T>C mutation in Fras1bfb/bfb mice. (B) Sequence alignment of the Fras1 protein sequence showing the amino acid residue affected in Fras1bfb/bfb is highly conserved across evolution. (C) Schematic of the Fras1 protein detailing protein domains, location of Fras1bfb/bfb mutation in the C-terminal region, the blebs mutation (bl), and reported human mutations (NM_025074). ?heterozygous,+compound heterozygous, #homozygous, black; Fraser Syndrome, red; CAKUT, blue; Ablepharon macrostomia syndrome/Fraser Syndrome.
Table 1.
Phenotypes observed in Fras1bfb/bfb embryos.
Figure 3.
Immunofluoresence in E15.5 embryonic epidermis of the skin at the level of the kidneys.
Fras1 expression in Fras1+/+ (A) and Fras1bfb/bfb (B) shows mislocalisation of the protein to the basal membrane in mutant tissue (arrowheads). Rabbit IgG control (C). Western blot analysis indicates comparable levels of Fras1 protein in Fras1+/+, Fras1+/bfb and Fras1bfb/bfb samples (D and E).
Figure 4.
Morphology of the palate in Fras1+/+ (A,C,E and G) and Fras1bfb/bfb (B,D,F and H) mice.
At E18.5 the palate has fused completely in all wildtype foetuses (A) but a proportion of Fras1bfb/bfb mice have an overt palatal cleft (B). The palatal shelves have elevated and become closely opposed in wildtype mice (C) while there is a significant delay in palatal shelf growth in Fras1bfb/bfb embryos (D). At E15.5 there is a complete fusion between the palatal shelves and nasal septum in wildtype mice (E) while a subtle palatal blister is evident in 80% of E15.5 Fras1bfb/bfb embryos (arrowhead, F). TGFβ3 is strongly expressed in palatal shelf epithelium (arrow) of both wildtype (G) and Fras1bfb/bfb mutants (H) but is aberrantly expressed in the nasal septum epithelium (arrowhead) in mutants. NC, nasal cavity; NS, nasal septum; VN, vomeronasal organ; T, tongue; MC, Meckel's cartilage, PS, palatal shelf.
Figure 5.
Preaxial polydactyly and blood-filled blisters in the hindlimbs of Fras1bfb/bfb mice.
Blood-filled blisters are evident in the hindlimbs of Fras1bfb/bfb at E14.5 (A and B) and E17.5 (C and D), often distorting the digits. A proportion of Fras1bfb/bfb exhibit preaxial polydactyly (asterisks in E-H), evident in E14.5 and E17.5 wholemounts (E and F), E17.5 skeletal preparations (G) and E12.5 Sox9 in situ stained embryos (H). Evidence of a blister can also be seen in an E17.5 skeletal preparation (arrowhead in G). Digit numbers are indicated in (G).
Figure 6.
Sternum and kidneys are abnormal in Fras1bfb/bfb embryos.
Sternabrae are misaligned in Fras1bfb/bfb mice at E18.5 (B), when compared to the sternum of Fras1+/+ embryos of the same age (A). Analysis of the internal organs of Fras1+/+ (C and E) and Fras1bfb/bfb (D and F) mice show no identifiable kidneys in the majority Fras1bfb/bfb mutants (asterisk in D). A blood-filled blister is also present in the hindlimb of this embryo (arrowhead in D). Transverse sections of an E17.5 Fras1bfb/bfb embryo show a large void where the kidney should be located (asterisk in F). A, adrenal gland; K, kidneys.