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

Genotyping results for <i>Lmx1b^Icst/+</i> intercross.

a<p>Test for significance using Chi-square test.</p

Forepaw skeletal phenotype of transgenic rescue mice.

<p>µCT scans of the ventral and dorsal sides 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) 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 <i>Lmx1b^Icst</i>^/<i>Icst</i> rescue mice homozygous for the transgene and for the <i>Lmx1b^KO/KO</i> rescue hemizygous for the BAC transgene. Arrows point to sesamoid bones that are a feature of the ventral surface. In the case of the <i>Lmx1b^KO/KO</i> 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 <i>Lmx1b^KO/KO</i> hemizygous for the BAC transgene which was P26.</p

Homozygosity for the BAC transgene can rescue both the <i>Icst</i> and knockout lethal phenotypes.

<p>For each cross the progeny from two matings was counted.</p>a<p>all are homozygous for the transgenic BAC.</p>b<p>includes 1 taken for analysis before weaning.</p>c<p>includes 3 taken for analysis before weaning. There were 2 found dead on P0.</p>d<p>includes 5 taken for analysis before weaning. There were 4 that did not survive to weaning.</p>e<p>includes 2 taken for analysis before weaning. There was 1 found dead on P1.</p>f<p>includes 4 taken for analysis before weaning. There were 3 that that did not survive to weaning.</p>g<p>Test for significance using Chi-square test.</p

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

Survival of <i>Lmx1b^Icst/+</i> compared to wild-type at P1 in an <i>Lmx1b^Icst/+</i> x wild-type cross.

a<p>67 live pups observed at P0 missing at P1 (unknown genotype).</p>b<p>Test for significance using Chi-square test.</p

Genotyping results for backcross and intercross of the two <i>Lmx1b</i> alleles.

a<p>Test for significance using Chi-square test.</p>b<p>4 pups found dead at birth with ventralised limbs.</p><p>- = not possible genotypes.</p

<i>Icst</i> causes defects of the optic nerve and the ganglion cell layer of the retina.

<p>(A) Sections through the optic nerve of adult mice. The optic nerve is variably affected in <i>Lmx1b^Icst/+</i> mice. Wild-type (WT) is normal (upper panel, left). In some <i>Lmx1b^Icst/+</i> (<i>Icst</i>/+) 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 <i>Lmx1b^Icst/+</i> (<i>Icst</i>/+), <i>Lmx1b</i>^KO/<i>+</i> (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 <i>Lmx1b^Icst/+</i> and n = 2 for the other two genotypes). Compared to wild-type and <i>Lmx1b</i>^KO/<i>+</i> mice which show no difference (P = 0.95) the number of ganglion cells is greatly reduced in the <i>Lmx1b^Icst</i>^/<i>+</i> mice compared to wild-type (P = 0.017) and to <i>Lmx1b</i>^KO/<i>+</i> mice (P = 0.034). (C) Optic nerve damage in <i>Lmx1b^Icst/+</i> mice. Optic nerve damage was assessed in wild-type (WT) and <i>Lmx1b^Icst</i>^/<i>+</i> (<i>Icst</i>/+) mice at 8 months (WT, n = 23 and <i>Icst</i>/+, n = 16) and at 10–11 months (WT, n = 26 and <i>Icst</i>/+, 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004359#pgen.1004359-Anderson1" target="_blank">[59]</a>. 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).</p

Wild-type and <i>Icst</i> LMX1B are in a complex with LDB1.

<p>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.</p

<i>Icst</i> is a homeodomain missense mutation of <i>Lmx1b</i> that abolishes protein function.

<p>(A) Genomic DNA sequence traces from <i>Lmx1b</i> exon 5 from wild-type (WT), heterozygous mutant (<i>Icst</i>/+) and homozygous mutant (<i>Icst</i>/<i>Icst</i>) embryonic samples. The position of the T-to-A transversion at position 725 (725T>A) in the <i>Lmx1b</i> gene, numbering from Genbank entry AF078166, is highlighted. This transversion causes a V265D mutation in the homeodomain numbering from entry O88609 in <a href="http://www.uniprot.org" target="_blank">http://www.uniprot.org</a>. (B) The <i>Icst</i> mutation impairs binding of LMX1B. Bandshift analysis using a fixed amount of ³²P-labelled FLAT LMX1B binding element <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004359#pgen.1004359-Morello1" target="_blank">[21]</a> 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 <i>Icst</i> 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 <i>Icst</i> mutation. The position of the probe alone is also indicated by an arrow. (C) The <i>Icst</i> mutation abolishes transcriptional activity. A luciferase reporter plasmid, 6×FLAT-C2P-Luc4, containing six copies of the LMX1B binding element FLAT upstream of the <i>Col2a1</i> promoter (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004359#pgen.1004359-Morello1" target="_blank">[21]</a>, 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 <i>Icst</i> mutation (t-test; * = P<0.005).</p