Supplementary Information
Supplemental Figure 1
Undescended gonads in triple mutant XY embryos. SEM analysis of the genito-urinary system of E18.5
embryos. The left panel shows embryos of XX, and the right panel of XY genotype. Arrows indicate the
position of gonads in each panel, narrow arrowheads indicate the uterus, and wide arrowhead the bladder.
The gene mutation is indicated on the left. The wild type, Irr, Ir/Irr and Igf1r/Irr homozygous null
embryos were identical. Ovaries are adjacent to the kidney (a,c,e,g); and testes are caudal to the kidney and
adjacent to the bladder neck (b,d,f). Genotypically male embryos mutant for Ir/Igf1r/Irr have gonads
situated next to the kidney (h). The size of the triple mutant XY gonads (h) is comparable to that of the XX
gonad (g). Mice carrying a mutation in Igf1r and both Ir and Igf1r are reduced in size compared to their
littermates, as originally described5,6,29. The scale bar is 0.5mm. k, kidney.
Supplemental Figure 2
Karyotypic and molecular analysis of Y chromosome in triple mutant mice.
a)
Embryonic fibroblasts were used for chromosomal spreads. Left panel shows the total chromosomal complement,
middle panel shows the sex chromosomes, and right panel indicates whether gonads have descended.
b) Genotypic analysis of wild type, single, double and triple mutant embryos for genes expressed on the Y
chromosome. To test for the presence of an “intact” Y chromosome in the mutant embryos, several Y-specific
genes Sry, Zfy, and Ubely1 were amplified by PCR. -actin was used to ensure presence of DNA in each sample.
In wild type and Irr mutant embryos, presence of Y chromosome correlates with descended gonads, in Ir/Igf1r
double mutant XY embryos, gonads are partially descended and in Ir/Igf1r/Irr triple mutant XY embryos gonads
do not descend.
The following PCR primer pairs were used. Sry: (5’ CAG CCC TAC AGC CAC ATG AT 3’; 5’ GAG TAC AGG
TGT GCA GCT CTA 3’). Zfy (5’ CAG AAC CCT TTG GTA CAC TG 3’; 5’ AAC ACC ACT CTC AAG AGT AG
3’); Ubely1 (5’ GGA GAT TGA GAG AAG GCA CA 3’; 5’ GTC CAG TGT AGG CAA ACA CA 3’), -actin
(5’CCT AGG CAC CAG GGT GTG AT3’; TCA CGG TTG GCC TTA GGG TT3’).
Supplemental Figure 3
Histological analysis of the reproductive tract from single, double and triple mutant embryos. H&E stained low
magnification pictures of transverse sections of embryos at E17.5 are shown. Gonads are indicated by arrows. In
single and double mutant XX embryos ovaries are situated next to kidneys (a,c,e,g) and testes are situated next to
bladder (b,d,f). In triple mutant XY embryos (h), the gonads are adjacent to the kidneys. bl, bladder, k, kidney, sc,
spinal cord.
Supplemental Figure 4.
Mice lacking Ir/Irr/Igf1r display Müllerian duct structure at the histological and molecular levels. Sections
through single, double and triple mutant XX embryos show typical uterine morphology – the surrounding stroma and
the luminal epithelial lining (a,c,e,g). A distinct vas deferens with a narrow lumen is present in the single and double
mutant males (b,d,f), but not in Ir/Igf1r/Irr XY embryos, instead a uterus indistinguishable from wild type females is
present (h). ut, uterus, vd, vas deferens.
Molecular markers for uterus show the presence of Müllerian duct structures and absence of Wollfian duct structures
in the triple mutant XY embryos. In wild type, Wnt4, MisRII and Wnt7a are expressed only in the Müllerian duct
derivatives in the XX embryos and not in Wollfian duct derivatives in XY embryos. Wnt 4 and MisRII (i,j,k,l) are
expressed in the stroma region, whereas Wnt7a is expressed in the luminal epithelium (arrowhead, m,n). In the triple
mutant XY embryos expression of all three uterine markers is observed (j’,l’,n’). ut, uterus; vd, vas deferens.
Supplemental Figure 5
a) Cell proliferation is decreased in the triple mutant XY gonads at E12.5. The total number of BrdU-positive
cells in the control XY gonad is set at 100%. In the top 35m of the control XY gonad, the majority of BrdU-positive
cells are somatic cells, in contrast, in the female and the triple mutant XY gonad, significantly more germ cells are
observed. Cell proliferation in the triple mutant XY gonads compared with control XY gonad is reduced *(p< 0.03)
whereas the total number of germ cells in the mesenchyme underneath the coelomic epithelium is increased ***
(p<0.0001) and the number of proliferating somatic cells is decreased **(p<.01).
b) SYBR green real-time RT-PCR analysis for the Insulin family receptors. The relative level of Ir mRNA, Irr
mRNA and Igf1r mRNA in urogenital ridges at E10.5 to E13.5 were estimated by SYBR green real-time RT-PCR,
and normalized to the corresponding Gapdh levels. For each gene analyzed, the expression level in the female gonad
at E10.5 was arbitrarily set at 1. The values are means ± standard deviation. At each stage analyzed, expression levels
of all three insulin family receptors are statistically similar between the male and the female gonad. However, there is
a statistically significant increase in relative expression of each receptor between E11.5 and E12.5, and a statistically
significant decrease in the expression of Ir at E13.5 (p<0.005).
c)
Expression of insulin receptor family members in the developing gonad. RNA was isolated from individual
female and male gonads at E10.5, E11.5 and E12.5. -actin was used to normalize the amount of cDNA in each
gonad. Approximately equal amounts of cDNA from each age were used for amplification. Expression of all
three receptors is detected in both sexes between E10.5-E12.5. Adult kidney cDNA was used as a positive
control and no DNA was added to the negative control. +, positive control; -, negative control; F, female; M,
male; RT, reverse transcriptase.
Supplemental Table 1. Gonadal phenotypes in mutant animals
E16.5 or E17.5 mutant gonads were analyzed at histological (H&E staining) and molecular level (by ISH) to assess
gonadal phenotypes. Testes were defined at the morphological level by the presence of testicular cords and by
expression of male markers (Sox9, Mis, Insl3). On the other hand, ovotestes were defined by the presence of some
testicular cords and expression of male (Sox9, Mis, Insl3) and female markers (Fig) and ovaries by the absence of
testicular cords and male markers (Sox9, Mis, Insl3) but with expression of female marker (Fig).