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

Combinatorial targeting of early pathways profoundly inhibits neurodegeneration in a mouse model of glaucoma.

The endothelin system is implicated in various human and animal glaucomas. Targeting the endothelin system has great promise as a treatment for human glaucoma, but the cell types involved and the exact mechanisms of action are not clearly elucidated. Here, we report a detailed characterization of the endothelin system in specific cell types of the optic nerve head (ONH) during glaucoma in DBA/2J mice. First, we show that key components of the endothelin system are expressed in multiple cell types. We discover that endothelin 2 (EDN2) is expressed in astrocytes as well as microglia/monocytes in the ONH. The endothelin receptor type A (Ednra) is expressed in vascular endothelial cells, while the endothelin receptor type B (Ednrb) receptor is expressed in ONH astrocytes. Second, we show that Macitentan treatment protects from glaucoma. Macitentan is a novel, orally administered, dual endothelin receptor antagonist with greater affinity, efficacy and safety than previous antagonists. Finally, we test the combinatorial effect of targeting both the endothelin and complement systems as a treatment for glaucoma. Similar to endothelin, the complement system is implicated in a variety of human and animal glaucomas, and has great promise as a treatment target. We discovered that combined targeting of the endothelin (Bosentan) and complement (C1qa mutation) systems is profoundly protective. Remarkably, 80% of DBA/2J eyes subjected to this combined inhibition developed no detectable glaucoma. This opens an exciting new avenue for neuroprotection in glaucoma. Neurobiol Dis 2014 Aug 15; 71C:44-52

Generating embryonic stem cells from the inbred mouse strain DBA/2J, a model of glaucoma and other complex diseases.

Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background

Chimerism and germ line transmission of DBA/2J ES cells.

<p>Four DBA/2J ES cell lines (two derived from each media type, 1i and 3i) were assessed for capacity to contribute to the germ line. These data are based on standard microinjection of ES cells into C57BL/6J host blastocysts. Additional data, including laser assisted microinjection data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050081#pone.0050081.s001" target="_blank">Table S1</a>. Details are provided for: the number of chimeric mice (determined based on coat showing at least partial dilute brown agouti color, column 2); the % of those chimeric mice that were male (column 3); the % of male chimeric mice that sired offspring with 100% dilute brown agouti color (column 4); and the % of offspring that were completely dilute brown agouti (transmission rate, column 5).</p

DBA/2J ES cell-derived male chimeras.

<p>DBA/2J ES cells were injected into C57BL/6J-derived blastocyst. Estimated percentage of coat derived from DBA/2J ES cells (dilute brown agouti) was used to determine the percent chimerism in each pup.</p

DBA/2J ES cell derivation.

<p>(<b>A</b>) In each round of derivation, 11–60% of embryos gave rise to stable embryonic stem cell lines (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050081#pone-0050081-t001" target="_blank">Table 1</a>). (<b>B</b>) Approximately ∼20% of lines were male with normal chromosome counts, regardless of culture conditions. (C) All of the ES cell lines, regardless of culture conditions, expressed the essential pluripotency markers NANOG, SSEA-1 and OCT-3/4 (POU5F1).</p

Generating a conditional allele of <i>Edn2</i> directly in DBA/2J-derived ES cells.

<p>(<b>A</b>) A targeting construct was generated that included <i>loxP</i> sites flanking exon 2 (e2) of the <i>Edn2</i> gene. Exon 2 contains part of the key functional domain for Edn2, and removal of exon 2 induces a frameshift for protein translation (<b>B</b>) Correct targeting at the 5′ end of the Edn2 gene was assessed using Xba1 restriction digests and southern blotting. The presence of the Neomycin (NEO) selectable marker incorporates an additional <i>Xba</i>1 restriction enzyme site in the targeted allele, generating a 6.6 kb fragment (Flox) compared to the normal 8.7 kb fragment (WT). Black arrows indicate clones correctly targeted at the 5′ end. (<b>C</b>) An example of a chimeric mouse generated as a result of injection of targeted DBA/2J ES cell clones into C57BL/6J blastocysts. (<b>D–E</b>) Chimeric male mice were mated with DBA/2J females and offspring genotyped for the heterozygous presence of the loxP sites in intron 1 (WT = 481 bp, Flox = 631 bp, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050081#s2" target="_blank">Methods</a>). White arrows indicate mice that carry the floxed allele of <i>Edn2</i>. (D). See methods for primer information. As expected, 100% of heterozygous mutant offspring had the DBA/2J coat color (dilute brown agouti, E).</p

Variable NANOG expression in DBA/2J ES cell lines.

<p>(<b>A–D</b>) Representative images and FLOW analysis of 3i (AC175, A,C) and 1i lines (AC216, B,D) immunolabeled with an antibody to NANOG. Robust NANOG expression was characteristic of lines derived and cultured in 3i conditions.</p

Radiation treatment inhibits monocyte entry into the optic nerve head and prevents neuronal damage in a mouse model of glaucoma

Glaucoma is a common ocular disorder that is a leading cause of blindness worldwide. It is characterized by the dysfunction and loss of retinal ganglion cells (RGCs). Although many studies have implicated various molecules in glaucoma, no mechanism has been shown to be responsible for the earliest detectable damage to RGCs and their axons in the optic nerve. Here, we show that the leukocyte transendothelial migration pathway is activated in the optic nerve head at the earliest stages of disease in an inherited mouse model of glaucoma. This resulted in proinflammatory monocytes entering the optic nerve prior to detectable neuronal damage. A 1-time x-ray treatment prevented monocyte entry and subsequent glaucomatous damage. A single x-ray treatment of an individual eye in young mice provided that eye with long-term protection from glaucoma but had no effect on the contralateral eye. Localized radiation treatment prevented detectable neuronal damage and dysfunction in treated eyes, despite the continued presence of other glaucomatous stresses and signaling pathways. Injection of endothelin-2, a damaging mediator produced by the monocytes, into irradiated eyes, combined with the other glaucomatous stresses, restored neural damage with a topography characteristic of glaucoma. Together, these data support a model of glaucomatous damage involving monocyte entry into the optic nerve

Forepaw phenotype of transgenic rescue mice.

<p>Pictures of the ventral and dorsal sides of forepaws from wild-type (WT), <i>Lmx1b^Icst</i>^/<i>Icst</i> (<i>Icst</i>/<i>Icst</i>) and <i>Lmx1b^KO/KO</i> (KO/KO) mice are shown and whether the BAC transgene is hemizygous or homozygous is indicated (BACx1 or BACx2). The ventral side of the paw is normal for all the rescue mice. The dorsal surface of all the homozygous mutant paws appears ventralised with pigmented footpads and no hair. The ages of the mice shown are as follows. Top two rows, P14; third row P26 and bottom row P35.</p