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1.
Figure 8

Figure 8. Pax2 immunofluoresence on COS-7 cells transfected with wild-type or mutant Pax2 expression vectors.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

Both wild-type and mutant proteins display nuclear localization, as evidenced by co-localization of green fluorescence (Pax2) with DAPI nuclear staining (blue).

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
2.
Figure 6

Figure 6. Comparison of Pax2 mRNA steady-state levels and Pax2 protein stability in wild-type and mutant expression vector-transfected NIH/3T3 cells.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

Although steady-state levels of Pax2 mRNA were comparable in wild-type and mutant transfected cells (A), the short-term protein stability of mutant Pax2 protein products were considerably reduced compared to wild-type, as determined in cycloheximide translation-inhibition experiments (B,C). See also Table 1 for quantification of mRNA levels.

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
3.
Figure 7

Figure 7. Comparison of wild-type and mutant Pax2 expression in embryonic mouse tissue.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

Pax2 immunofluorescence on parasagittal sections of E11.5 wild-type and homozygous mutant embryos demonstrate a normal pattern of expression in the ventral optic stalk (A). The level of Pax2 expression, however, is qualitatively reduced in the mutant mice. This reduced steady-state level of expression was confirmed by Western blot in heterozygous and homozygous mutant embryos (B).

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
4.
Figure 3

Figure 3. Histologic sections of Pax2+/+ and Pax2A220G/A220G mouse eyes at three embryonic time points.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

At E11.5, parasagittal sections reveal a delay in apposition of the edges of the optic fissure in mutant mice (arrow) (A,B). At E13.5, coronal sections through the wild-type and homozygous mutant embryos reveal a delay in the formation of the tunica vasculosis lentis (arrow) (C,D). At E17.5, parasagittal sections demonstrate non-fusion of the optic fissure (uveal coloboma) in mutant embryos (arrow) (E,F). V = ventral retina.

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
5.
Figure 4

Figure 4. Histologic sections of Pax2+/+ and Pax2A220G/A220G mouse kidneys (axial) and cerebellum (sagittal) at E17.5.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

Whereas wild-type mice have begun to develop renal glomeruli (arrow, A) and tubules (arrowhead, A), the mutant mice have only primordial kidneys with poor differentiation of these structures (arrow, B) In contrast, the differentiation of the cerebellum of both wild-type (C) and mutant (D) mice is comparable at this time, despite the midbrain-hindbrain boundary being a site of Pax2 expression during embryogenesis. By E14.5, cranial structure was grossly normal in both wild-type (E) and homozygous mutant (F) embryos.

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
6.
Figure 5

Figure 5. Comparison of wild-type and mutant Pax2 protein transactivation and expression in cell culture.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

NIH/3T3 cells were transfected with expression constructs for wild-type or mutant Pax2 along with a Pax2-responsive luciferase reporter gene. All three mutants tested show reduced ability to transactivate (A). When steady-state levels of Pax2 protein were compared on Western blots from these experiments, mutants showed consistently lower levels of expression (B). Similar findings were observed when these experiments were replicated in COS-7 cells (data not shown).

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
7.
Figure 9

Figure 9. Electrophoretic mobility shift assay comparing DNA binding of wild-type and three mutant Pax2 proteins.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

A labeled Pax2 DNA-binding consensus sequence was incubated in the presence or absence of nuclear extract of COS-7 cells expressing equal amounts of the wild-type or mutant Pax2 protein; the same, unlabeled, competing DNA oligonucleotide; and/or a mutated version of the unlabeled oligonucleotide (Mut-Pax2). Nuclear extracts from mock transfected cells did not appreciably result in a shift of the labeled Pax2 DNA-binding site oligonucleotide, whereas wild-type and all three mutant Pax2 proteins bound the oligonucleotide with approximately equal affinity. Specificity for this binding was shown by competing the binding with the same, unlabeled oligonucleotide sequence and by failure of an unlabeled mutant oligonucleotide to compete for binding.

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
8.
Figure 1

Figure 1. Clinical ocular phenotype in C57BL/6-Pax2+/A220G mice compared to wild-type, C57BL/6 mice.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

(A) Fundus photograph of C57BL/6 mouse showing normal optic nerve and radial pattern of retinal blood vessels. (B) Fundus photograph of C57BL/6-Pax2+/A220G mouse showing congenital excavation of the optic nerve head with peripapillary pigment changes (arrow). (C) Lectin immunofluorescence of wild-type C57BL/6 mouse showing normal, radial vessel patterning. (D,E) Lectin immunofluorescence of C57BL/6-Pax2+/A220G mice showing abnormal vascular patterning, including curving of vessels towards the dorsal retina (D, arrows, d = dorsal, v = ventral) and separation of the central retinal vascular trunks (E, arrows). Histologic section of a Pax2+/+ (F) and a Pax2+/A220G (G) mouse eye through the optic nerve and peripapillary retina showing abnormal excavation of the optic nerve (G, arrow) and retinal rosette formation (G, arrowhead). Remnants of the tunica vasculosis lentis and mild extension of the retinal pigment epithelium were variably noted in histopathology from other Pax2A220G/+ eyes (data not shown).

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.
9.
Figure 2

Figure 2. Homology modeling of the wild-type and mutant Pax2 paired domain-DNA complex.. From: Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human.

The paired domain of wild-type Pax2 domain DNA are represented by red and white ribbons, respectively, and their corresponding atomic structures are shown by red and white bonds (A). Hydrogen bonds are shown in blue. Threonine 74 in the mouse protein sequence (equivalent to T75A in human) is absolutely conserved across several species (B) and across all known murine Pax-family members (C). Fragments of the Pax2 paired domain–DNA complex modified by the mutations T74A, dup73ET and G75S are shown on (D–F), respectively. Hydrogen bonds presented in the wild type protein that are broken by the mutation T74A are labeled as 1 and 2 for (D) and by the mutation G75S is labeled as 3 (F). Yellow arrows indicate the location of mutations in Pax2 paired domain. A schematic of the Pax2b protein modified from Lechner et al. [32] showing the paired domain (gray), the octapeptide (Oct) domain (yellow), and the C-terminus, which is rich in proline, serine, threonine and tyrosine (PSTY) residues (G). Numbers indicate amino acid position. The arrow denotes the approximate position of the three mutations studied.

Ramakrishna P. Alur, et al. PLoS Genet. 2010 March;6(3):e1000870.

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