Urine analysis of line <i>Pou3f3</i>^<i>L423P</i> (in an additional group of mice not transferred to the German Mouse Clinic).

<p>Urine analysis of line <i>Pou3f3</i>^<i>L423P</i> (in an additional group of mice not transferred to the German Mouse Clinic).</p

Generation and Standardized, Systemic Phenotypic Analysis of <i>Pou3f3^L423P</i> Mutant Mice

<div><p>Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (<i>Pou3f3</i>) gene, which leads to the amino acid exchange <i>Pou3f3</i>^<i>L423P</i> thereby affecting the conserved homeobox domain of the protein. <i>Pou3f3</i> homozygous knockout mice are published and show perinatal death. Line <i>Pou3f3</i>^<i>L423P</i> is a viable mouse model harboring a homozygous <i>Pou3f3</i> mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of <i>Pou3f3</i>^<i>L423P</i> homozygous mutants identified significantly regulated genes as compared to wild-type controls.</p></div

Immunohistochemical localization of POU3F3 in kidneys.

<p>POU3F3 was detected in nuclei of numerous but not all tubule segments with weak up to strong staining intensity in a wild-type kidney. The immunostaining pattern and staining intensity of POU3F3 in the kidney of a <i>Pou3f3</i>^<i>L423P</i> homozygous mutant mouse was similar to the findings observed in the wild-type control kidney. DAB (brown color); nuclear staining: haemalum (blue color). Age of mice analyzed: 22 months. Bars represent 100 μm.</p

Neurology analysis by modified SHIRPA of line <i>Pou3f3</i>^<i>L423P</i>.

<p>Neurology analysis by modified SHIRPA of line <i>Pou3f3</i>^<i>L423P</i>.</p

Morphological analysis of the inner ears of line <i>Pou3f3</i>^<i>L423P</i>.

<p>Morphological analysis of the inner ears using the whole mount clearing method showed abnormal superior semi-circular canal formation in the homozygous mutants of line <i>Pou3f3</i>^<i>L423P</i>. Left picture shows a control inner ear with normal semi-circular canals. Right picture shows the smaller inner ear of a <i>Pou3f3</i>^<i>L423P</i> homozygous mutant animal with abnormal superior semi-circular canal formation. Red arrow shows the constriction of the superior semi-circular canal. SSCC: superior semi-circular canal, PSCC: posterior semi-circular canal, ASCC: anterior semi-circular canal.</p

Clinical chemical analysis of line <i>Pou3f3</i>^<i>L423P</i>.

<p>Clinical chemical analysis of line <i>Pou3f3</i>^<i>L423P</i>.</p

Analysis of <i>Pou3f3</i> in wild-type and <i>Pou3f3</i>^<i>L423P</i> mutant mice.

<p>(A) Electropherogram of the <i>Pou3f3</i>^<i>L423P</i> mutation. The box shows the T→C point mutation at nt 1268 (ENSMUST00000054883) from the wild-type codon leucine (L) to the mutant codon proline (P) at amino acid position 423. (B) Genotyping of mice by allele-specific PCR-RFLP reaction. <i>Sml</i>I restriction digest of the 460 bp PCR product results in 353 bp and 107 bp fragments of the wild-type allele. Hom, <i>Pou3f3</i>^<i>L423P</i> homozygous mutant; Het, <i>Pou3f3</i>^<i>L423P</i> heterozygous mutant; Wt, wild-type; M, pUC Mix 8 marker, MBI Fermentas. (C) Partial protein sequence alignment of the homeobox domain of murine POU3F3 with other species. The underlined amino acid residue shows the position of the <i>Pou3f3</i>^<i>L423P</i> mutation.</p

Open field behavioral analysis of line <i>Pou3f3^L423P</i>.

<p>Open field behavioral analysis of line <i>Pou3f3^L423P</i>.</p

Dual energy X-ray absorption (DXA) analysis of bone- and weight-related parameters in line <i>Pou3f3</i>^<i>L423P</i>.

<p>Dual energy X-ray absorption (DXA) analysis of bone- and weight-related parameters in line <i>Pou3f3</i>^<i>L423P</i>.</p

Analysis of energy metabolism in line <i>Pou3f3</i>^<i>L423P</i>.

<p>Analysis of energy metabolism in line <i>Pou3f3</i>^<i>L423P</i>.</p