Characterisation of XY <i>Fgfr2^hob/hob</i> embryonic gonad development on the C57BL/6J (B6) background and complementation test with the <i>Fgfr2^tm1.1Dor</i> null allele.

<p>A) WMISH analysis of gonads at 14.5 dpc from XY wild-type, XX wild-type and XY <i>Fgfr2^hob/hob</i> embryos using a marker of the Sertoli cell lineage (<i>Sox9</i>), ovarian somatic cells (<i>Wnt4</i>) and meiotic germ cells (<i>Stra8</i>). B) Embryos homozygous for the <i>Fgfr2^tm1.1Dor</i> allele (<i>Dor/Dor</i>) 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 <i>Fgfr2^hob</i> and <i>Fgfr2^tm1.1Dor</i> alleles (<i>Dor/hob</i>) lack limbs and are noticeably smaller than wild-type controls (+/+). D) Upper panel: <i>Sox9</i> WMISH of 13.5 dpc embryonic gonads from control and XY <i>Fgfr2^tm1.1Dor/hob</i> doubly heterozygous embryos; lower panel: <i>Stra8</i> 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.</p

Quantitation of phospho-p38 MAPK (p-p38) levels in gonadal samples at 11.5 dpc (16–18 ts) in XY wild-type and <i>Fgfr2^hob/hob</i> gonads.

<p>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.</p

Normal <i>Sry</i> expression, but disrupted <i>Sox9</i> expression, in XY <i>Fgfr2^hob/hob</i> embryonic gonads.

<p>A) <i>Sry</i> WMISH at 11.5 dpc (16 ts) showing expression in XY wild-type (left) and XY <i>Fgfr2^hob/hob</i> (right) gonads. B, C) anti-SRY immunostaining at 18 ts in wild-type (B) and XY <i>Fgfr2^hob/hob</i> (C) gonads. D) <i>Sox9</i> 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 <i>Fgfr2^hob/hob</i> (F) gonads. G) <i>Sox9</i> WMISH at 23 ts in XY wild-type (left) and XY <i>Fgfr2^hob/hob</i> (right) gonads. H, I) anti-SOX9 immunostaining at 23 ts in XY wild-type (H) and XY <i>Fgfr2^hob/hob</i> (I) gonads. J) <i>Sox9</i> WMISH at 12.5 dpc in XY wild-type (left) and XY <i>Fgfr2^hob/hob</i> (right) gonads. K, L) anti-SOX9 immunostaining at 13.0 dpc in wild-type (K) and XY <i>Fgfr2^hob/hob</i> (L) gonads.</p

Complete XY gonadal sex reversal in <i>Fgfr2^hob/hob</i> embryos on B6.

<p>A–C) Immunostaining with anti-AMH antibody of gonadal sections from XY wild-type (A), XX wild-type (B) and XY <i>Fgfr2^hob/hob</i> (C) embryos at 12.5 dpc. D–F) anti-FOXL2 immunostaining of samples equivalent to those in A–C. G–I) WMISH for <i>Insl3</i> (a marker of Leydig cells) of gonads with same genotypes as A–C. J–M) <i>Oct4</i> WMISH of 11.5 dpc (17 ts) gonads from control XY (J), XX (K), XY <i>Fgfr2^hob/hob</i> and XX <i>Fgfr2^hob/hob</i> gonads. N–Q) <i>Oct4</i> WMISH of 13.5 dpc gonads from embryos of the same genotype as J-M.</p

The hobbyhorse (<i>hob</i>) mutation disrupts XY sex determination and is caused by an ENU-induced point mutation of <i>Fgfr2</i>.

<p>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 <i>in situ</i> hybridisation (WMISH) with a <i>Sox9</i> 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 <i>Fgfr2</i> of a hobbyhorse embryo. Upper trace is wild-type, lower trace is hobbyhorse. E) The proline residue that is mutated in the <i>hob</i> allele is highly conserved in vertebrates. Mm, <i>Mus musculus</i>; Hs, <i>Homo sapiens</i>; Gg, <i>Gallus gallus</i>; Xl, <i>Xenopus leavis</i>; Dr, <i>Danio rerio</i>. F) Diagrammatic representation of FGFR2 and its domain structure in the FGFR2b and FGFR2c isoforms. The <i>hob</i> mutation (asterisk) resides in the third extracellular immunoglobulin-like domain, encoded by the invariant exon 7.</p

Tomato expression in E18.5 <i>Fgf10^iCre/+; Tomato^flox/+</i> lungs.

<p>Recombination was induced at E15.5 by a single IP injection of tamoxifen. Note the absence of Tomato expression in <i>Fgf10^+/+; Tomato^flox/+</i> lungs (<b>A; </b><i>n = 3</i>). The regions in the dotted boxes are magnified in (<b>B</b>) and (<b>C</b>). Tomato-positive cells are observed in the lung mesenchyme and interlobular septae of <i>Fgf10^iCre/+; Tomato^flox/+</i> lungs (white arrows) (<b>A’, B’; </b><i>n = 3</i>). Inset in B’ shows high magnification of interlobular septae. Labeled cells in the trachea arrange in ring-like structures (black arrows) (<b>C’; </b><i>n = 3</i>). (<b>A”-C”; </b><i>n = 8</i>) X-Gal staining of <i>Fgf10^LacZ/+</i> lungs at E18.5. Inset in B” shows high magnification of interlobular septae. Tam: tamoxifen.</p

Bioinformatic screening for putative regulatory elements 3 kb downstream of <i>Fgf10</i> ATG.

<p>(<b>A</b>) The screening reveals regions that are rich in transcription factor binding sites conserved between mouse and human (as predicted by rVista; <a href="http://rvista.dcode.org" target="_blank">http://rvista.dcode.org</a>). Lung-related transcription factor binding sites within the 3′ end of the deleted sequence are shown. (<b>B</b>) Screening for lung-related H3K4me3 modification sites (as predicted by UCSC Genome Browser; <a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>). A dense H3K4me3 modification site is predicted in the region overlapping exon 1-intron 1 boundary of <i>Fgf10</i> gene. UTR: Untranslated region; ECRs: Evolutionary conserved regions.</p

Inducible conditional <i>Fgf10</i> inactivation using <i>Fgf10^iCre</i> driver line.

<p>Cre was activated by tamoxifen food from E8.5 to E14.5 in <i>Fgf10^iCre/flox; Tomato^flox/+</i> embryos. Labeled cells are present in the limbs, lung and cecum (<b>D, G, H, L</b>). Note the webbing of the digits in the forelimbs (arrows indicate webbing sites and dashed lines indicate peripheries) (<b>H vs. E, M</b>) and the hypomorph-like phenotype in the cecum (<b>L vs... </b><b>I, K</b>). <i>Fgf10^iCre/flox; Tomato^flox/+</i> lungs show a deformed shape (<b>... </b><b>vs... </b><b>B</b>) as well as branching simplification (<b>G vs... </b><b>F, J</b>). Dashed lines indicate the epithelium in the lung (<b>F, G</b>) and cecum (<b>I, L</b>). <i>n = 3</i>. Data are shown as average values ± SEM. * <i>P</i><0.05; **** <i>P</i><0.0001; Tam: tamoxifen.</p

Tomato expression in E18.5 <i>Fgf10^iCre/+; Tomato^flox/+</i> embryos.

<p>Recombination was induced at E15.5 by a single IP injection of tamoxifen. Note the absence of Tomato expression in <i>Fgf10^+/+; Tomato^flox/+</i> embryos (<b>A–D</b>). Tomato-positive cells are detected in the ear, skin, limbs and cecum (<b>A’–D’</b>). (<b>A”–D”)</b> Higher magnifications of dotted boxes in <b>A’, B’, C’, D’</b>. <i>n = 3</i>. Tam: tamoxifen.</p

Signaling induced by FGFR2b-ligands interactioncontrols progressive limb growth along the proximal-distal axis.

<p>Pregnant females carrying [<i>R26^rtTA/+;Tg/+</i>] double transgenic (DTG) embryos and single transgenic [<i>R26^rtTA/+ or Tg/+</i>] control embryos were treated continuously with Doxycycline food starting at different developmental stages; (<b>A,B</b>) Treatment at E8.5, before limb induction: loss of both hindlimbs and forelimbs in E13.5 DTG embryos. (<b>C,D</b>) Treatment at E10.5, after limb bud induction: Formation of rudimentary forelimbs and almost complete absence of hindlimbs in E14.5 DTG embryos. (<b>E–F</b>) Treatment at E11.5: Absence of autopod in both hindlimbs and forelimbs of E13.5 DTG embryos. (<b>G</b>) Dissected hindlimbs in DTG and controls shown in (E,F). (<b>H–I</b>) Treatment at E13.0: control (H) and DTG (I) embryos at E16. Note that the <i>Topgal</i> allele was introduced in DTG and control embryos to visualize the extent of mesenchymal condensation in the limb. (J,K) Dissected left hindlimbs from embryos shown in H and I displaying failure of separation of the digits in DTG hindlimb. (<b>L–O</b>) Treatment at E13.5: truncation of the digits in both forelimbs and hindlimbs. (<b>P–S</b>) Alcian blue/alizarin red staining indicates the reduction in the size of the P3 phalange in the forelimb and complete loss of the P3 phalange in the hindlimb of DTG embryos treated from E13.5 to E16.5. d, digits; p, phalanges.</p