The <i>bfb</i> mouse harbours a mutation in Fras1.

<p>(A) Chromatogram of <i>Fras1</i> gene sequence identifying the c.10762T&gt;C mutation in <i>Fras1^bfb/bfb</i> mice. (B) Sequence alignment of the Fras1 protein sequence showing the amino acid residue affected in <i>Fras1^bfb/bfb</i> is highly conserved across evolution. (C) Schematic of the Fras1 protein detailing protein domains, location of <i>Fras1^bfb/bfb</i> mutation in the C-terminal region, the <i>blebs</i> mutation (<i>bl</i>), and reported human mutations (NM_025074). ^?heterozygous,⁺compound heterozygous, ^#homozygous, black; Fraser Syndrome, red; CAKUT, blue; Ablepharon macrostomia syndrome/Fraser Syndrome.</p

Extracellular matrix products in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.

<p>Collagen IV (A–D) and fibronectin (E–H) staining sections from NS- (A, C, E &amp; G) and ADR-injected (B, D, F &amp; H) wild type (A, B, E &amp; F) and eNOS-deficient (C, D, G &amp; H) kidneys at day 28. Graph showing quantification of the area of staining for collagen IV and fibronectin. <i>One-way ANOVA</i>, n = 5, data are means ± SD. ***: <i>vs</i> WT NS, WT ADR and eNOS KO NS, <i>P</i>&lt;0.001.</p

Glomerular endothelial cell and podocyte damage in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.

<p>Time course of glomerular endothelial cell CD31 (A–E) and podocyte synaptopodin (F–J) staining sections from NS-treated kidneys at day 28 (A&amp;F), ADR-treated kidneys at days 3 (B&amp;G), 7 (C&amp;H), 14 (D&amp;I) and 28 (E&amp;J). Graph showing quantification of the area of CD31(K) and synaptopodin (L) staining. <i>One-way ANOVA</i>, n = 5, data are means ± SD. <i>Vs</i> NS day 28, * <i>P</i>&lt;0.05; **<i>P</i>&lt;0.01; ***<i>P</i>&lt;0.001.</p

Pathological characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.

<p>PAS staining of sections from NS (A&amp;C) and ADR-injected (B&amp;D) wild type (A&amp;B) and eNOS-deficient (C&amp;D) mice at day 28. Masson trichrome staining of sections from NS (E&amp;G) and ADR-injected (F&amp;H) wild type (E&amp;F) and eNOS-deficient (G&amp;H) mice at day 28. eNOS-deficient mice with ADR-induced nephropathy exhibited well developed exudative (fibrin-cap) lesions, glomerular sclerosis, interstitial fibrosis and inflammation at day 28. Original magnifications, 400 X.</p

Morphology of the palate in <i>Fras1^+/+</i> (A,C,E and G) and <i>Fras1^bfb/bfb</i> (B,D,F and H) mice.

<p>At E18.5 the palate has fused completely in all wildtype foetuses (A) but a proportion of <i>Fras1^bfb/bfb</i> 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 <i>Fras1^bfb/bfb</i> 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 <i>Fras1^bfb/bfb</i> embryos (arrowhead, F). <i>TGFβ3</i> is strongly expressed in palatal shelf epithelium (arrow) of both wildtype (G) and <i>Fras1^bfb/bfb</i> 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.</p

Functional characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.

<p><b>A:</b> Ratio of urinary protein/creatinine; <b>B:</b> Body weight; <b>C:</b> Ratio of kidney /body weight; <b>D:</b> Serum creatinine and <b>E:</b> Systolic blood pressure in NS- and ADR-injected mice. <i>Two-way ANOVA</i>; n = 5, data are means ± SD.</p

Preaxial polydactyly and blood-filled blisters in the hindlimbs of <i>Fras1^bfb/bfb</i> mice.

<p>Blood-filled blisters are evident in the hindlimbs of <i>Fras1^bfb/bfb</i> at E14.5 (A and B) and E17.5 (C and D), often distorting the digits. A proportion of <i>Fras1^bfb/bfb</i> 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).</p

Sternum and kidneys are abnormal in <i>Fras1^bfb/bfb</i> embryos.

<p>Sternabrae are misaligned in <i>Fras1^bfb/bfb</i> mice at E18.5 (B), when compared to the sternum of <i>Fras1^+/+</i> embryos of the same age (A). Analysis of the internal organs of <i>Fras1^+/+</i> (C and E) and <i>Fras1^bfb/bfb</i> (D and F) mice show no identifiable kidneys in the majority <i>Fras1^bfb/bfb</i> 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 <i>Fras1^bfb/bfb</i> embryo show a large void where the kidney should be located (asterisk in F). A, adrenal gland; K, kidneys.</p

Immunofluoresence in E15.5 embryonic epidermis of the skin at the level of the kidneys.

<p>Fras1 expression in <i>Fras1^+/+</i> (A) and <i>Fras1^bfb/bfb</i> (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 <i>Fras1^+/+</i>, <i>Fras1^+/bfb</i> and <i>Fras1^bfb/bfb</i> samples (D and E).</p

Representative <i>bfb</i> phenotypes at developmental age E11.5 (A), E13.5 (B), E15.5 (C) and E17.5 (D).

<p>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).</p