Mutation of the diamond-blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes

Abstract

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.

Figure 1. Heterozygous mutation of Rps7 results in visible white spotting, small body size, and tail kinking.

(A) Montu (Mtu) heterozygous mice exhibiting a white belly spot and kinked tail were identified in an ENU mutagenesis screen. An independent ENU screen identified zuma (Zma) mice with similar phenotypes (data not shown). (B) Heterozygote Mtu/+ male (blue) and female (green) mice have a significantly reduced body weight compared to wild-type male (gray) and female (red) littermates. (C) Sequencing revealed novel Rps7 point mutations in Mtu/+ (c.574T>G, encoding p.V156G) and Zma/+ (c.637A>C, encoding p.Y177S) mice. Sequence traces shown are for the antisense strand. Detailed Rps7 genomic structure information can be found at http://www.ncbi.nlm.nih.gov/gene/20115. (D) The predicted structural locations of mutated amino acids in Rps7^Mtu and Rps7^Zma alleles. The three-dimensional structures of RPS7 orthologs from S. cerevisiae (PDB ID 3U5C_H, red) and T. thermophila (PDB ID 2XZM_3, gray) are superimposed. The locations of the residues homologous to mouse p.V156 (*) and p.Y177 (arrow) are shown in green (S. cerevisiae) and in yellow (T. thermophila). Image generated with UCSF Chimera.

Figure 2. Rps7^Mtu shows reduced function in ribosomal precursor processing.

(A) Western blot showing similar levels of expression for N- and C-terminal FLAG-tagged wild-type RPS7 (WT-N and WT-C, respectively) and RPS7^Mtu (M-N and M-C, respectively) proteins in HEK-293 cells. The RPS7-specific band is indicated by *, and NPT2 (arrowhead) expression is shown as a control. (B) Subcellular localization of N- and C-terminal FLAG-tagged RPS7 proteins. Wild-type RPS7 and RPS7^Mtu both localize to speckles in the nucleus and are observed throughout the cytoplasm. Scale bar = 10 um. All panels are at the same magnification. (C) Representative Northern blot analysis of liver and brain RNA from wild-type (WT) and Rps7^Mtu/+ (M) mice detecting various rRNA precursors using a probe within the internal transcribed spacer (ITS1). The 30S and 21S rRNA precursors are indicated. (D) Quantitation of the signals of Northern experiments reported as the ratio between 30S and 21S rRNA precursors was significantly different between Rps7+/+ and Rps7^Mtu/+ (* indicates p<0.01). The average of the values is reported in the bar graphs with S.E.M.

Figure 3. Skeletal abnormalities in Rps7 mutants.

(A, B) Comparative X-rays of adult Rps7+/+ (A) and Rps7^Zma/+ (B) mice show severe vertebral fusion leading to tail kinking in an Rps7^Zma/+ mutant. (C–H) Alcian blue and alizarin red skeletal staining of late gestation Rps7+/+ (C, E, G) and Rps7^Zma/+ (D, F, H) embryos. (C, D) Disorganization within the neural arches of Rps7^Zma/+ is evident in a dorsal view of the cervical vertebrae. (E, F) Asymmetric attachment of 8 ribs to the sternum (arrow) and a shortened first sternebrae (*) are indicated in Rps7^Zma/+. (G, H) Delayed development and disorganization of the ossification centers is apparent in the lumbar and sacral vertebrae. The sacral vertebrae show abnormal fusing of additional vertebrae (bracketed region). Scale bars: C,D,G,H = 2 mm; E,F = 200 µm.

Figure 4. Eye dysmorphology in Rps7 mutants.

Representative whole-mount images from one Rps7+/+ and two different Rps7^Zma/+ embryos are shown at E12.5 (A–C) and 14.5 (D–F). In addition, H&E stained sagittal sections through the eye are shown for one Rps7+/+ and two different Rps7^Zma/+ embryos at E18.5 (G–I). The eye dysmorphology of Rps7^Zma/+mutants ranges in severity from minor unilateral or bilateral uveal coloboma (E, H) to severe microphthalmia resulting in disorganized eye structures (C, F, I). Arrows in E and F mark examples of coloboma and extreme microphthalmia, respectively. Arrow in H marks abnormal folding of the retinal layers. All images are oriented with anterior up, rostral to the right. Within each age group/row, all genotypes are shown at the same magnification. Scale bars = 0.5 mm.

Figure 5. Peripheral-blood parameters appear normal with slight developmental delay in Rps7^Zma/+ fetal liver red cell precursors.

(A) Rps7^Mtu/+ mice displayed similar complete blood count (CBC) values to their wild-type littermates with the exception of a very slightly elevated mean corpuscular volume (MCV) (* indicates p<0.05; N = 7). (B) Rps7^Zma/+ mice did not differ significantly from their wild-type littermates in any CBC measurements (N = 5). (C) A typical example of FACS analysis of fetal liver cells. The 5 characterized erythroid cell populations, R1–R5, are boxed in pink. (D) The average values and standard deviation for each fetal liver erythroid cell population are given as a percentage of total viable cells in Rps7+/+ and Rps7^Zma/+ samples (* indicates p<0.001; N = 6).

Figure 6. Melanoblast numbers are reduced in Rps7^Zma/+ mutants.

(A, B) In transgenic embryos carrying the melanoblast reporter Tg(Dct-LacZ), whole mount staining showed that Dct-positive melanoblasts are significantly reduced at E10.5 in Rps7^Zma/+; Tg(Dct-LacZ) mice (A) compared to Rps7+/+;Tg(Dct-LacZ) littermates (B). The reduction is noticeably apparent over the otic region (red circle). (C,D) Whole mount staining of E14.5 Rps7^Zma/+; Tg(Dct-LacZ) embryos showed that these embryos (D) also display a reduction in melanoblasts relative to Rps7+/+;Tg(Dct-LacZ) littermates (C). The microphthalmia observed in Rps7^Zma/+ mice is apparent in (D). (E, F) Consistent with the whole-mount observations, transverse vibratome sections through the trunk of E14.5 embryos revealed very few melanoblasts in the developing skin of Rps7^Zma/+; Tg(Dct-LacZ) mice (F) as compared to the numerous melanoblasts seen in Rps7+/+;Tg(Dct-LacZ) littermates (arrow, E). Blue punctate staining indicates positive signal in melanoblasts. In all pairs of images, Rps7+/+ and Rps7^Zma/+ are at the same magnification. NT = neural tube.

Figure 7. Brain size and behavioral abnormalities in Rps7 mutants.

(A) A Nissl stained coronal section of a 5 month old Rps7^Mtu/+brain shows a thinner cortex and larger ventricles when compared to an Rps7+/+ littermate. (B, C) Dissected whole brains show that the cortex is reduced in size in adult Rps7^Mtu/+ (B) and postnatal day 0 Rps7^Zma/+mice (C) when compared to Rps7+/+ littermates. (D–I) Magnetic Resonance Microscopy (MRM) was used to visualize brain development in late gestation (E18.5) Rps7+/+(D–F) and Rps7^Zma/+(G–I) embryos. Enlarged ventricles (v in panel H) were apparent in all Rps7^Zma/+ samples (N = 3). Representative slices are shown in sagittal (D,G), coronal (E,H), and axial (F,I) views. Scale bars = 1 mm. (J) The volume of each brain region was quantitated as a percentage of total brain volume in Rps7+/+(black columns) and Rps7^Zma/+(gray columns) (N = 3). Abbreviations: Olfactory bulbs (OB), lateral ventricles (LV), cortex (Ct), septum (Sp), striatum (St), 4^th ventricle (4V), hippocampus (Hp), thalamus (Th), colliculi (Co), cerebellum (Ce). ** indicates p<0.005. (K) Assessment of working memory by measuring spontaneous alternation in a T-maze showed a significant difference between Rps7^Mtu/+mice and Rps7+/+ littermate controls (* indicates P = 0.01, N = 9). In all panels +/+ = Rps7+/+; Mtu/+ = Rps7^Mtu/+; Zma/+ = Rps7^Zma/+.

Figure 8. Increased apoptosis occurs in the developing CNS of Rps7^Zma/+ mutant embryos; however, this apoptosis is reduced in Rps7^Zma/+; Trp53^KO/+ embryos.

(A–C) Increased apoptosis was observed in E11.5 Rps7^Zma/+ (Z/+) coronal sections through the neocortex compared to Rps7+/+ (+/+) and Rps7^Zma/+; Trp53^KO/+ (Z/+;p53/+), as measured by cleaved caspase-3 (CC3) staining shown in green. (D–F) Apoptosis was also relatively increased in neural tube cross-sections of Rps7^Zma/+ embryos at E11.5 (E). (G–I) Cellular disorganization was apparent in mitotic, phospho-histone H3-positive (PH3+) cells surrounding the lumen of the neural tube in E11.5 Rps7^Zma/+ embryos (H). (J) E11.5 Rps7^Zma/+ (z) embryos showed no difference from Rps7+/+(+) or Rps7^Zma/+; Trp53^KO/+(p) in total counts of PH3+ cells surrounding the lumen of the neural tube. (K) CC3+ cell counts confirmed significantly increased apoptosis in E11.5 Rps7^Zma/+ (z) neural tube as compared to Rps7+/+ (+) or Rps7^Zma/+; Trp53^KO/+ (p) (* indicates p<0.001). Scale bars: in A, D, G = 100 µM with equivalent magnification across all genotypes.