Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

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.This research was supported in part by the Intramural Research Program of NHGRI, NIH, and the Wellcome Trust and by NHMRC Australia grant 366746. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Epilepsy in Dcx Knockout Mice Associated with Discrete Lamination Defects and Enhanced Excitability in the Hippocampus

Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence, were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to further study how lamination defects underlie the genesis of epileptiform activities

Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human

Branching and nucleokinesis defects in migrating interneurons derived from doublecortin knockout mice

International audienceType I lissencephaly results from mutations in the doublecortin (DCX) and LIS1 genes. We generated Dcx knockout mice to further understand the pathophysiological mechanisms associated with this cortical malformation. Dcx is expressed in migrating interneurons in developing human and mouse brains. Video microscopy analyses of such tangentially migrating neuron populations derived from the medial ganglionic eminence show defects in migratory dynamics. Specifically, the formation and division of growth cones, leading to the production of new branches, are more frequent in knockout cells, although branches are less stable. Dcx-deficient cells thus migrate in a disorganized manner, extending and retracting short branches and making less long-distant movements of the nucleus. Despite these differences, migratory speeds and distances remain similar to wild-type cells. These novel data thus highlight a role for Dcx, a microtubule-associated protein enriched at the leading edge in the branching and nucleokinesis of migrating interneurons

Brain size and behavioral abnormalities in <i>Rps7</i> mutants.

<p>(A) A Nissl stained coronal section of a 5 month old <i>Rps7^Mtu/+</i>brain shows a thinner cortex and larger ventricles when compared to an <i>Rps7</i>+/+ littermate. (B, C) Dissected whole brains show that the cortex is reduced in size in adult <i>Rps7^Mtu/+</i> (B) and postnatal day 0 <i>Rps7^Zma/+</i>mice (C) when compared to <i>Rps7</i>+/+ littermates. (D–I) Magnetic Resonance Microscopy (MRM) was used to visualize brain development in late gestation (E18.5) <i>Rps7</i>+/+(D–F) and <i>Rps7^Zma/+</i>(G–I) embryos. Enlarged ventricles (v in panel H) were apparent in all <i>Rps7^Zma/+</i> 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 <i>Rps7</i>+/+(black columns) and <i>Rps7^Zma/+</i>(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 <i>Rps7^Mtu/+</i>mice and <i>Rps7</i>+/+ littermate controls (* indicates P = 0.01, N = 9). In all panels +/+ = <i>Rps7</i>+/+; Mtu/+ = <i>Rps7^Mtu/+</i>; Zma/+ = <i>Rps7^Zma/+</i>.</p

Melanoblast numbers are reduced in <i>Rps7^Zma/+</i> mutants.

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