Supplementary Table 1. Numbers and percentages of normal and malformed
embryos collected from homozygous Wif1lacZ/lacZ mice at different embryonic
days
Figure S1. Homozygous Wif1lacZ/lacZ embryos display developmental defects. In
normally developed homozygous Wif1lacZ/lacZ embryos (Normal), development of
frontonasal processes are prominent at E12.5; digits of hindlimb (HL) are well formed
by E14.5. In about 5% of homozygous Wif1lacZ/lacZ embryos (Malformed),
development of frontonasal processes, fore and hindlimb bud are retarded at E12.5
(n=3). By E14.5, limb development is severely retarded.
Abbreviations: fn, frontal
nasal; flb, fore limbbud; hlb, hindlimb bud.
Figure S2. X-gal staining of Wif1lacZ/lacZ embryos from E12.5-E14.5. LacZ
expression is under the control of the native promoter of Wif1, and patterns of -gal
expression reflect endogenous pattern of Wif1 expression. X-gal staining was detected
in the forebrain, frontalnasal region, fore and hind limb buds, and developing GT (A,
B, F, G), in the apical mesenchyme of the genital tubercle (C, H), in the midline apical
urs endoderm and in the cloaca membrane endoderm (D, E, I, J) at E12.5 and E13.5. By
E14.5, the LacZ expression was decreased in the forebrain, limb and ribs but high level
of expression can still be found in the interdigit region of the developing limbs (K, L).
Strong expression of LacZ could still be detected in the agm, the urethral plate (M, N)
and no expression was detected in the endoderm in ud (O). Black arrows indicated
LacZ expression in the midline cloaca epithelium. Abbreviations: agm, apical genital
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mesenchyme; cl, cloaca; cm, cloaca membrane; re, rectum; lb, limb bud; pf, preputial
fold; ud, urethral duct; urs, urorectal septum; up, urethral plate.
Figure S3. Ectopic Wif1 expression in ETU-treated ARMs embryos. Wif1 (brown)
was expressed in the apical urs endoderm and proximal cm at E14.5 in control rat
embryos (A). Expression of Wif1 was found in the urethral duct endoderm and the
urogenital sinus endoderm at E15.5 (B). Upon treatment of ETU, rat embryos
displayed urorectal defects with unseptated cloaca at E14.5 and hypospadic-like
phenotypes at E15.5 (C, D). Wif1 was ectopically expressed in the endoderm of the
urs and the rectum in both E14.5 and E15.5 ETU-treated rat embryos. Scale bar: 50
m. Abbreviations: cl, cloaca; cm, cloaca membrane; gt, genital tubercle; re, rectum;
ugs, urogenital sinus; urs, urorectal septum; ud, urethral duct.
Figure S4. Wif1 expression of urorectal endoderm is not affected by
Dorsomorphin. GT explant from E12 embryos were cultured for 24 hours without
(Ctrl) or with Dorsomorphin. The urs has descended approaching the cloaca
membrane in control explant (A). In contrast, descent of the urs was drastically
retarded in dorsomorphin-cultured explant (B). Wif1 expression (brown) was detected
in the urorectal endoderm of control (A) and dorsomorphin-cultured (B) GT explants.
Phosphorylated Smad1 (brown) was frequently detected in the urorectal endoderm of
control explant (C; arrowheads), but not in dorsomorphin-treated explant (D). Scale
bar: 50 m. Abbreviations: urs, urorectal septum; cl, cloaca; cm, cloaca membrane.
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