Figure 1.
The hobbyhorse (hob) mutation disrupts XY sex determination and is caused by an ENU-induced point mutation of Fgfr2.
A) A wild-type XY gonad (left) showing characteristic testicular morphology at 14.5 dpc, in contrast to two XY hobbyhorse mutants identified in a forward genetic screen, which have disrupted testis cords (centre) or lack cords entirely (right). All gonads shown are after wholemount in situ hybridisation (WMISH) with a Sox9 probe. B) A hobbyhorse mutant (right) lacks limbs. A wild-type embryo is also shown (left). C) Absence of lung development in a hobbyhorse embryo (right), in contrast to normal lungs at the same stage (left). D) Sequence trace showing homozygosity for a C to T mutation (asterisk) in exon 7 of Fgfr2 of a hobbyhorse embryo. Upper trace is wild-type, lower trace is hobbyhorse. E) The proline residue that is mutated in the hob allele is highly conserved in vertebrates. Mm, Mus musculus; Hs, Homo sapiens; Gg, Gallus gallus; Xl, Xenopus leavis; Dr, Danio rerio. F) Diagrammatic representation of FGFR2 and its domain structure in the FGFR2b and FGFR2c isoforms. The hob mutation (asterisk) resides in the third extracellular immunoglobulin-like domain, encoded by the invariant exon 7.
Figure 2.
Characterisation of XY Fgfr2hob/hob embryonic gonad development on the C57BL/6J (B6) background and complementation test with the Fgfr2tm1.1Dor null allele.
A) WMISH analysis of gonads at 14.5 dpc from XY wild-type, XX wild-type and XY Fgfr2hob/hob embryos using a marker of the Sertoli cell lineage (Sox9), ovarian somatic cells (Wnt4) and meiotic germ cells (Stra8). B) Embryos homozygous for the Fgfr2tm1.1Dor allele (Dor/Dor) are much smaller than wild-type controls (+/+) at 11.5 dpc and also lack limbs. C) Embryos at 14.5 dpc doubly heterozygous for the Fgfr2hob and Fgfr2tm1.1Dor alleles (Dor/hob) lack limbs and are noticeably smaller than wild-type controls (+/+). D) Upper panel: Sox9 WMISH of 13.5 dpc embryonic gonads from control and XY Fgfr2tm1.1Dor/hob doubly heterozygous embryos; lower panel: Stra8 WMISH of 14.5 gonads from embryos of same genotypes as upper panel. The developmental stage of the doubly heterozygous gonad in the lower panel appears significantly retarded when compared to the XX control.
Figure 3.
Complete XY gonadal sex reversal in Fgfr2hob/hob embryos on B6.
A–C) Immunostaining with anti-AMH antibody of gonadal sections from XY wild-type (A), XX wild-type (B) and XY Fgfr2hob/hob (C) embryos at 12.5 dpc. D–F) anti-FOXL2 immunostaining of samples equivalent to those in A–C. G–I) WMISH for Insl3 (a marker of Leydig cells) of gonads with same genotypes as A–C. J–M) Oct4 WMISH of 11.5 dpc (17 ts) gonads from control XY (J), XX (K), XY Fgfr2hob/hob and XX Fgfr2hob/hob gonads. N–Q) Oct4 WMISH of 13.5 dpc gonads from embryos of the same genotype as J-M.
Figure 4.
Normal Sry expression, but disrupted Sox9 expression, in XY Fgfr2hob/hob embryonic gonads.
A) Sry WMISH at 11.5 dpc (16 ts) showing expression in XY wild-type (left) and XY Fgfr2hob/hob (right) gonads. B, C) anti-SRY immunostaining at 18 ts in wild-type (B) and XY Fgfr2hob/hob (C) gonads. D) Sox9 WMISH at 18 ts with tissue samples as described in (A). E, F) anti-SOX9 immunostaining at 18 ts in XY wild-type (E) and XY Fgfr2hob/hob (F) gonads. G) Sox9 WMISH at 23 ts in XY wild-type (left) and XY Fgfr2hob/hob (right) gonads. H, I) anti-SOX9 immunostaining at 23 ts in XY wild-type (H) and XY Fgfr2hob/hob (I) gonads. J) Sox9 WMISH at 12.5 dpc in XY wild-type (left) and XY Fgfr2hob/hob (right) gonads. K, L) anti-SOX9 immunostaining at 13.0 dpc in wild-type (K) and XY Fgfr2hob/hob (L) gonads.
Figure 5.
Quantitation of phospho-p38 MAPK (p-p38) levels in gonadal samples at 11.5 dpc (16–18 ts) in XY wild-type and Fgfr2hob/hob gonads.
A) Lane view images showing Simple Western detection of p-p38, p38, and α-tubulin. B) Graph showing the ratio of p-p38 to tubulin in the two gonadal genotypes. The ratio of p-p38 to p38 was similarly unaltered. Errors were calculated using standard error mean.