A dominant-negative mutation of mouse Lmx1b causes glaucoma and is semi-lethal via LDB1-mediated dimerization [corrected]

Abstract

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.

Figure 1. Icst is a homeodomain missense mutation of Lmx1b that abolishes protein function.

(A) Genomic DNA sequence traces from Lmx1b exon 5 from wild-type (WT), heterozygous mutant (Icst/+) and homozygous mutant (Icst/Icst) embryonic samples. The position of the T-to-A transversion at position 725 (725T>A) in the Lmx1b gene, numbering from Genbank entry AF078166, is highlighted. This transversion causes a V265D mutation in the homeodomain numbering from entry O88609 in http://www.uniprot.org. (B) The Icst mutation impairs binding of LMX1B. Bandshift analysis using a fixed amount of ³²P-labelled FLAT LMX1B binding element with increasing amounts (as indicated by the triangles) of His-tagged fusion proteins containing the homeodomain or full-length LMX1B either wild-type (WT) or containing the Icst mutation (ICST). In lane 1 no protein was added (-). Complexes as indicated by the arrows were formed with the wild-type proteins but not the proteins containing the Icst mutation. The position of the probe alone is also indicated by an arrow. (C) The Icst mutation abolishes transcriptional activity. A luciferase reporter plasmid, 6×FLAT-C2P-Luc4, containing six copies of the LMX1B binding element FLAT upstream of the Col2a1 promoter (, kind gift of Brendan Lee), was either transfected alone (-) or co-transfected with empty pcDNA-3.1 vector (V) or pcDNA-3.1 containing the wild-type (WT-L) or Icst (Icst-L) long form of LMX1B or wild-type (WT-S) or Icst (Icst-S) short form of LMX1B. Each experiment was performed in triplicate, and mean firefly luciferase expression normalized to control renilla luciferase expression is shown+standard error. Both WT-L and WT-S elicit robust induction of luciferase expression that reduced to background levels by the Icst mutation (t-test; * = P<0.005).

Figure 2. Icst causes defects in the anterior segment of the eye.

(A) Lmx1b^KO/+ (KO/+) eye that is of normal appearance. (B) Lmx1b^Icst/+ (Icst/+) eye where there is a fine strand between the pupillary margin and the cornea (arrowed). (C) This Lmx1b^Icst/+ eye is enlarged with severe corneal vascularisation and central opacity. (D–F). Histological appearance of corneas. (D) Wild-type normal cornea (WT). (E) Lmx1b^Icst/+ cornea that has stromal vascularisation (barbed arrows). Descemet's membrane is wrinkled (dashed arrows) and a membrane is present on the anterior surface of the iris (arrowheads). (F) Lmx1b^Icst/+ cornea with basal epithelial oedema (dashed arrow). A stromal blood vessel with red blood cells is indicated by a barbed arrow. (G) Normal wild-type iridocorneal angle. (H–J) Iridocorneal angle morphology of Lmx1b^Icst heterozygotes display a spectrum of phenotypes ranging from open-angle (H) to closed-angle morphology where the iris can be seen adhering to the cornea and blocking the iridocorneal angle as indicated by bars (I, J). (K–L) Higher magnification of wild-type (K) and Lmx1b^Icst/+ (L) iridocorneal angles. (K) The wild-type has an open-angle with robust drainage structures ([). Schlemm's canal (arrowheads) is clearly identifiable and the trabecular meshwork (asterisks) has well-formed trabecular beams. (L) The Lmx1b^Icst/+ iridocorneal angle is open but narrow. Iridocorneal strands (IS) are present. These are a normal feature of the mouse angle but are not present at all locations and do not prevent aqueous humor drainage. The drainage structures are not as robust as in the WT ([). Schlemm's canal is present but compressed (arrowheads) and the trabecular meshwork is hypoplastic (asterisks). Original magnification ×40 (D and F) and ×20 (E). Scale bar = 50 µm.

Figure 3. The IOP is elevated in Lmx1b^Icst/+ mice.

(A) Box plot of IOP measurements of wild-type (WT) and Lmx1b^Icst/+ (Het) mice taken at different ages. There is significantly elevated IOP in the Lmx1b^Icst/+ mice up to 6 months (P = 0.006 at 2–3 months and P = 0.009 at 5–6 months). The IOP measurements are not significantly different in older mice owing to low readings of <10 mmHg in increasing numbers of the Lmx1b^Icst/+ mice. (B) Frequency histogram summarizing the data from panel A showing increased incidence of high IOP measurements in Lmx1b^Icst/+ mice.

Figure 4. Icst causes defects of the optic nerve and the ganglion cell layer of the retina.

(A) Sections through the optic nerve of adult mice. The optic nerve is variably affected in Lmx1b^Icst/+ mice. Wild-type (WT) is normal (upper panel, left). In some Lmx1b^Icst/+ (Icst/+) mice the optic nerve appears normal (lower panel, left) but in others there is evidence of optic nerve damage and cupping, indicated by asterisks (upper and lower panels, right). Scale bar = 50 µm. (B) Box plot of ganglion cell number present in Lmx1b^Icst/+ (Icst/+), Lmx1b ^KO/+ (KO/+) and wild-type (WT) mice. Ganglion cells were stained using an anti-BRN3 antibody and the number present in each of four areas from around the optic disc from one eye were counted (n = 3 for Lmx1b^Icst/+ and n = 2 for the other two genotypes). Compared to wild-type and Lmx1b ^KO/+ mice which show no difference (P = 0.95) the number of ganglion cells is greatly reduced in the Lmx1b^{Icst /+} mice compared to wild-type (P = 0.017) and to Lmx1b ^KO/+ mice (P = 0.034). (C) Optic nerve damage in Lmx1b^Icst/+ mice. Optic nerve damage was assessed in wild-type (WT) and Lmx1b^{Icst /+} (Icst/+) mice at 8 months (WT, n = 23 and Icst/+, n = 16) and at 10–11 months (WT, n = 26 and Icst/+, n = 36). Nerves with no glaucoma have axon counts that match controls (green bar). Nerves with moderate nerve damage have an average of 30% axon (yellow bar). This degree of damage occurs in a low percentage of wild-type of this strain background at this age . All severely affected nerves have extensive axon damage throughout the optic nerve with obvious axon loss ranging from 50% to almost complete axon loss (red bar).

Figure 5. Ultrastructural analysis of E17.5 and E18.5 kidneys shows that Icst causes glomerular defects when heterozygous.

(A–B) Wild-type (WT) and Lmx1b^KO/+ (KO/+) podocytes are normal with normal foot process formation (arrowheads). (C–D) Lmx1b^KO/KO (KO/KO) and Lmx1b^Icst/Icst (Icst/Icst) podocytes fail to form foot processes and appear immature. (E–L) Lmx1b^Icst heterozygous podocytes (Icst/+). (E) Normal foot process formation (arrowheads). (F) The bar indicates an area of the GBM that appears fragmented. (G–H) Foot processes (arrowheads) abut GBM that is split (arrows). (I) The podocytes appear cuboidal (as indicated by asterisks) and immature. (J) The GBM is split as indicated by the bar and foot processes are rudimentary (arrows). (K) There is a large split in the GBM that is filled with fibrillar material (as indicated by an asterisk). (L) Podocytes are positioned flush against a split GBM. P, podocyte; endo = endothelial cell. Scale bar = 2 µm.

Figure 6. Forepaw phenotype of transgenic rescue mice.

Pictures of the ventral and dorsal sides of forepaws from wild-type (WT), Lmx1b^{Icst /Icst} (Icst/Icst) and Lmx1b^KO/KO (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.

Figure 7. Forepaw skeletal phenotype of transgenic rescue mice.

µCT scans of the ventral and dorsal sides from wild-type (WT), Lmx1b^{Icst /Icst} (Icst/Icst) and Lmx1b^KO/KO (KO/KO) forepaws are shown and whether the BAC transgene is hemizygous or homozygous is indicated (BACx1 or BACx2). The ventral side of the skeleton appears normal for all the rescue mice (top panels). The dorsal surface appears completely ventralised for the Lmx1b^{Icst /Icst} rescue mice homozygous for the transgene and for the Lmx1b^KO/KO rescue hemizygous for the BAC transgene. Arrows point to sesamoid bones that are a feature of the ventral surface. In the case of the Lmx1b^KO/KO homozygous for the BAC transgene the dorsal surface appears more normal although there are still aspects of ventral morphology seen, for example a sesamoid bone (arrowed). The images shown are from mice between four and five weeks of age except for Lmx1b^KO/KO hemizygous for the BAC transgene which was P26.

Figure 8. Patella and scapula phenotype of transgenic rescue mice.

All mice shown are homozygous for the transgene. µCT scans of the patella and scapula are shown. (A) Lmx1b^{Icst /+} (Icst/+) with normal patella (arrowed). (B) Lmx1b^{Icst /Icst} rescue (Icst/Icst) knee where the patella is absent (arrow indicates its expected location). (C) Lmx1b^KO/KO rescue (KO/KO) knee where the patella is present (arrowed). (D) Normal scapula in Lmx1b^{Icst /+} (arrowed). (E) The scapula is very underdeveloped and small in Lmx1b^{Icst /Icst} rescue (arrowed). (F) The scapula appears more normal in Lmx1b^KO/KO rescue (arrowed). The ages of the mice shown are as follows; P23 (A, B, D, E), three months (C) and five weeks (F).

Figure 9. Wild-type and Icst LMX1B are in a complex with LDB1.

Western blot analysis of co-immunoprecipitation experiments using anti-Myc (α-Myc), anti-FLAG (α-FLAG) and anti-LDB1 (α-LDB1) antibodies. (A) Protein lysates (INPUT) of HEK 293T cells transfected with WT-Myc and ICST-FLAG as indicated were immunoprecipitated using anti-c-Myc antibody coupled to agarose and the bound fraction (BOUND) eluted. WT-Myc is present in the immunoprecipitated bound fraction but Icst-FLAG is not. (B) Protein lysates (INPUT) of HEK 293T cells transfected with pcDNA-LDB1 along with WT-Myc and ICST-FLAG as indicated were immunoprecipitated using anti-c-Myc agarose and the bound fraction (BOUND) eluted. Both LDB1 and ICST-FLAG are in a complex with the WT-Myc and found in the immunoprecipitated bound fraction.