A Dominant-Negative Mutation of Mouse Lmx1b Causes Glaucoma and Is Semi-lethal via LBD1-Mediated Dimerisation

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice

Spinster Homolog 2 (Spns2) Deficiency Causes Early Onset Progressive Hearing Loss

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing

A CNS-specific hypomorphic Pdgfr-beta mutant model of diabetic retinopathy

PURPOSE: A mouse mutant identified during a recessive N-ethyl-N-nitrosourea (ENU) mutagenesis screen exhibited ocular hemorrhaging resulting in a blood-filled orbit, and hence was named "redeye." We aimed to identify the causal mutation in redeye, and evaluate it as a model for diabetic retinopathy (DR). METHODS: The causative gene mutation in redeye was identified by haplotype mapping followed by exome sequencing. Glucose tolerance tests, detailed histologic and immunofluorescence analyses, and vascular permeability assays were performed to determine the affect of redeye on glucose metabolism, pericyte recruitment, and the development of the retinal vasculature and blood-retinal barrier (BRB). RESULTS: A mutation was identified in the Pdgfrb gene at position +2 of intron 6. We show that this change causes partial loss of normal splicing resulting in a frameshift and premature termination, and, therefore, a substantial reduction in normal Pdgfrb transcript. The animals exhibit defective pericyte recruitment restricted to the central nervous system (CNS) causing basement membrane and vascular patterning defects, impaired vascular permeability, and aberrant BRB development, resulting in vascular leakage and retinal ganglion cell apoptosis. Despite exhibiting classic features of diabetic retinopathy, redeye glucose tolerance is normal. CONCLUSIONS: The Pdgfrb(redeye/redeye) mice exhibit all of the features of nonproliferative DR, including retinal neurodegeneration. In addition, the perinatal onset of the CNS-specific vascular phenotype negates the need to age animals or manage diabetic complications in other organs. Therefore, they are a more useful model for diseases involving pericyte deficiencies, such as DR, than those currently being used

Endothelial FAK is required for tumour angiogenesis

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signalling and is an important player in cell migration and proliferation, processes vital for angiogenesis. However, the role of FAK in adult pathological angiogenesis is unknown. We have generated endothelial-specific tamoxifen-inducible FAK knockout mice by crossing FAK-floxed (FAKfl/fl) mice with the platelet derived growth factor b (Pdgfb)-iCreER mice. Tamoxifen-treatment of Pdgfb-iCreER;FAKfl/fl mice results in FAK deletion in adult endothelial cells (ECs) without any adverse effects. Importantly however, endothelial FAK-deletion in adult mice inhibited tumour growth and reduced tumour angiogenesis. Furthermore, in in vivo angiogenic assays FAK deletion impairs vascular endothelial growth factor (VEGF)-induced neovascularization. In addition, in vitro deletion of FAK in ECs resulted in reduced VEGF-stimulated Akt phosphorylation and correlating reduced cellular proliferation as well as increased cell death. Our data suggest that FAK is required for adult pathological angiogenesis and validates FAK as a possible target for anti-angiogenic therapies

ABR thresholds and SEM assessment suggest a local function of Spns2 in the inner ear.

<p>ABR thresholds (means +/−SD) are shown for homozygotes (red), heterozygotes (blue) and wildtypes (green), aged 7–14 weeks. Mice homozygous for the <i>Spns2^tm1a</i> allele displayed elevated ABR thresholds and degeneration of hair cells (<b><i>A</i></b><i> left</i>, <i>4 wks and </i><b><i>B</i></b>). By crossing with mice expressing Flp recombinase to excise the inserted cassette, <i>Spns2^tm1c/tm1c</i> mice were produced, which had normal ABR thresholds and normal hair cell morphology (<b><i>A</i></b><i> middle</i>, <i>8 wks and </i><b><i>C</i></b>). Then <i>Spns2^tm1c/tm1c</i> were crossed with <i>Sox10-Cre</i> mice to produce <i>Spns2^tm1d/tm1d</i>;Sox10-Cre mice which showed no response up to 95 dB SPL and hair cell degeneration with bulges and holes in the reticular lamina (<b><i>A</i></b><i> right</i>, <i>4 wks and </i><b><i>D</i></b>). SEM images are taken from the middle turn (40–70%) of the cochlea. Scale bar: 10 µm in <b><i>B,C,D</i></b>.</p

ABR thresholds of mice with <i>Spns2</i> conditionally inactivated in different tissues.

<p>ABR thresholds of individual mice are shown. All Cre driver lines showed normal thresholds (left column, in black. n = number of wildtype; number of mice carrying Cre). Most control littermates had normal responses (middle column: heterozygotes in blue; wildtypes in green). Homozygous <i>Spns2^tm1d</i> mutants (red) carrying the relevant Cre alleles are shown in the right column. <i>Spns2^tm1d</i> homozygotes carrying the Sox10-Cre allele had raised thresholds (top right), but the other four Cre lines had normal thresholds. The bottom row shows equivalent threshold data for the <i>Spns2^tm1c</i> allele and the Flp recombinase line used to generate this allele, again showing normal thresholds. There were three exceptions of individuals with raised thresholds (one heterozygote each in Pf4-Cre cross and Tie1-Cre cross, one homozygote in Tie1-Cre cross), which we believe probably carry an independent mutation causing the impairment (subject to ongoing positional cloning study).</p

Progressive deterioration of hair cells shown by scanning electron microscopy.

<p><i>Spns2^tm1a/tm1a</i> mice have a normal structure of hair bundles compared with wildtype mice at P21 (<b><i>A,B</i></b>). Scattered or patchy outer hair cell (OHC) degeneration was observed in the middle turn at P28 (<b><i>C,D</i></b>). Most stereocilia of OHCs in basal and middle turn have degenerated and stereocilia of IHCs were fused or lost at P56 (<b><i>E,F</i></b>). All the images were taken from the middle turn of the cochlea, defined as 40–70% of the distance along the cochlear duct from the base. Scale bar: 10 µm.</p

Eye defects in <i>Spns2^tm1a/tm1a</i> mice.

<p><b><i>A–C</i></b>, Phenotype screening was performed on 15 week old <i>Spns2^tm1a/tm1a</i> mice and identified buphthalmos, corneal opacity with vascularization and possible ulceration (<b><i>B</i></b>) and corneal opacity with vascularisation and polyp, thick discharge, elongated pupil which did not fully dilate with tropicamide (<b><i>C</i></b>). Albino mice were selected to achieve clearer demonstration, and a wildtype is shown in (<b><i>A</i></b>). <b><i>D</i></b>, Pupil-optic nerve section of a 15 week old wildtype eye. <b><i>E</i></b>, The <i>Spns2^tm1a/tm1a</i> mice showed a grossly abnormal eye with corneal opacity, vascularization, collapsed anterior chamber, small cataractous lens, and focal retinal degeneration. c = cornea, ac = anterior chamber, pc = posterior chamber, on = optic nerve. Scale bar = 450 µm. <b><i>F,G</i></b>,<b><i>H,I</i></b> Analysis of retinal vasculature was performed on retinal wholemounts from P10 pups (<b><i>F,G</i></b>) and 8 week old adult mice(<b><i>H,I</i></b>). Retinal vasculature was stained with Isolectin B4 (green) to visualise the endothelium and Proteoglycan NG2 (red) to visualise pericytes. Whereas arteries (a) appeared morphologically normal, veins (v) appeared thinner in <i>Spns2^tm1a/tm1a</i> (<b><i>I</i></b>) than in <i>Spns2^+/tm1a</i> (<b><i>H</i></b>) and had an irregular caliber with regions of narrowing (arrows). Although the retina has three capillary plexi only the primary plexus is shown at both P10 and 8 weeks as this is the first plexus to form and mature. Scalebars: 50 µm. <b><i>J</i></b>, Branch point analysis was performed on P10 retinal vasculature (<b><i>F,G</i></b>) to determine whether retinal vasculature showed any developmental abnormalities in vascular patterning. No significant difference was detected between <i>Spns2^tm1a/tm1a</i> (red) and <i>Spns2^+/tm1a</i> (blue) mice in the central retina (mature vessels) or the periphery, where the vessels are still developing at P10, in either the arteries or veins (Mann-Whitney U test for central branch points, p = 0.69; t-test for peripheral branch points, arterial p = 0.899, venous p = 0.996).</p

The hearing loss of <i>Spns2^tm1a/tm1a</i>, <i>Spns2^tm1b/tm1b</i> and <i>Spns2^tm1d/tm1d</i>; <i>Sox10-Cre</i> mice showed a similar pattern of progression.

<p>ABR thresholds of individual homozygotes (red), heterozygotes (blue) and wildtypes (green) are shown at 2, 3 and 14 or 4–10 weeks old. The control mice had immature thresholds at 2 weeks old and continued to mature to normal hearing levels at 3 weeks old. <i>Spns2^tm1a/tm1a</i> and <i>Spns2^tm1d/tm1d</i>; <i>Sox10-Cre</i> mice displayed progressive hearing loss from 2 to 3 weeks old. The red line in the middle bottom panel represents a control mouse homozygous for <i>Spns2^tm1c</i> but without carrying <i>Sox10-Cre</i>, and thus had no conditional knockout of <i>Spns2</i> in the inner ear and normal hearing.</p