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

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

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

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

Additional file 1: of Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair

The following additional data are available with the online version of this paper. Figure S1. In vitro assessment of sgRNA efficacy. Table S1. The sequences of the oligonucleotides used in this study. Table S2. The sequences and locations of the predicted off-target sites for the two sgRNAs used in design 1. Table S3. Oligonucleotide sequences for Sanger sequencing of sgRNA_U1 and sgRNA_D1 predicted off-target sites (three or fewer mismatches). (DOCX 922 kb

Pharmacological Inhibition of FTO

<div><p>In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function <i>in vitro</i> and <i>in vivo</i> as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate <i>in vitro</i>. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO’s demethylase activity could be therapeutically useful for the treatment of obesity.</p></div

Chemical structure of IOX3 and IC₅₀ values for FTO and PHD2.

<p>Chemical structure of IOX3 and IC₅₀ values for FTO and PHD2.</p

Oxygen Consumption Rate (OCR), Extracellular Acidification Rate (ECAR) of C2C12, and wild-type and FTO knockout MEFs treated with 1 μM IOX3 or an equivalent amount of vehicle control for 16 hours.

<p>A) OCR and, B) basal ECAR measured in C2C12 cells treated with vehicle (n = 10) and 1 μM IOX3 (n = 10) at baseline and after Oligomycin, FCCP and Rotenone treatment, data normalised to live stain. C) OCR and, D] ECAR measured in <i>Fto</i>^<i>+/+</i> and <i>Fto</i>^<i>-/-</i> MEFs cells treated with vehicle and 1 μM IOX3 (n = 5 per group) at baseline and after Oligomycin, FCCP and Rotenone treatment, data normalised to live stain. Data were analysed using a 2 way ANOVA with Bonferroni post-hoc test. Data is of readings following each compound injection and are expressed as mean ± SEM. E) Expression of FTO, phosphorylated-AMPKα and HIF-1α with representative ACTIN in cells treated with vehicle, 1uM IOX3, control scrambled siRNA or <i>Fto</i> siRNA for 24 hours. N = 3 biological replicates per condition.</p

Loss of KATNAL1 function leads to reduced numbers of stable microtubules in SCs.

<p>(a) Western blot analysis and (b) quantification of stable (glu) and dynamic (tyr) α-tubulin on d22 testes from wild-type and homozygous mutant animals reveals a significant reduction in numbers of stable microtubules in testes from <i>Katnal1^1H/1H</i> mutants (n = 5–7). (c) Immunohistochemical localisation of glu-α-tubulin on corresponding d22 testis sections localises stable microtubules to SC cytoplasm (arrows) and developing spermatids and in Wild-type animals. Conversely stable microtubules are below detection limits in much of the SC cytoplasm in homozygous mutant testes. (WT1 = Wilms Tumour 1, SC-specific loading control).</p