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

Comparative analysis of vertebrates reveals that mouse primordial oocytes do not contain a Balbiani body

Oocytes spend the majority of their lifetime in a primordial state. The cellular and molecular biology of primordial oocytes is largely unexplored; yet, it is necessary to study them to understand the mechanisms through which oocytes maintain cellular fitness for decades, and why they eventually fail with age. Here, we develop enabling methods for live-imaging-based comparative characterization of Xenopus, mouse and human primordial oocytes. We show that primordial oocytes in all three vertebrate species contain active mitochondria, Golgi and lysosomes. We further demonstrate that human and Xenopus oocytes have a Balbiani body characterized by a dense accumulation of mitochondria in their cytoplasm. However, despite previous reports, we did not find a Balbiani body in mouse oocytes. Instead, we demonstrate that what was previously used as a marker for the Balbiani body in mouse primordial oocytes is in fact a ring-shaped Golgi that is not functionally associated with oocyte dormancy. This study provides the first insights into the organization of the cytoplasm in mammalian primordial oocytes, and clarifies the relative advantages and limitations of choosing different model organisms for studying oocyte dormancy.This study was supported by a European Research Council Starting Grant (ERC-StG-2017-759107) and the Ministerio de Ciencia e Innovación (MINECO - BFU2017-89373-P and PID2020-115127GB-I00 to E.B.). G.Z. acknowledges funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 754422. M.S. is supported by a Juan de la Cierva-Formación fellowship from the Ministerio de Ciencia e Innovación (FJC2019-041607-I; AEI; 10.13039/501100011033)

A developmentally programmed splicing failure contributes to DNA damage response attenuation during mammalian zygotic genome activation

Transition from maternal to embryonic transcriptional control is crucial for embryogenesis. However, alternative splicing regulation during this process remains understudied. Using transcriptomic data from human, mouse, and cow preimplantation development, we show that the stage of zygotic genome activation (ZGA) exhibits the highest levels of exon skipping diversity reported for any cell or tissue type. Much of this exon skipping is temporary, leads to disruptive noncanonical isoforms, and occurs in genes enriched for DNA damage response in the three species. Two core spliceosomal components, Snrpb and Snrpd2, regulate these patterns. These genes have low maternal expression at ZGA and increase sharply thereafter. Microinjection of Snrpb/d2 messenger RNA into mouse zygotes reduces the levels of exon skipping at ZGA and leads to increased p53-mediated DNA damage response. We propose that mammalian embryos undergo an evolutionarily conserved, developmentally programmed splicing failure at ZGA that contributes to the attenuation of cellular responses to DNA damage.This work was funded by the Spanish Ministerio de Ciencia grants BFU2014-55076-P, BFU2017-89201-P and PID2020-115040GB-I00 (M.I.), Marie Skłodowska-Curie actions grant H2020-MSCA-IF-2014_ST-656843 (B.P.), La Caixa PhD fellowship (C.D.R.W.), and “Centro de Excelencia Severo Ochoa 2013-2017” SEV-2012-0208 (CRG-MI

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

Eye dysmorphology in <i>Rps7</i> mutants.

<p>Representative whole-mount images from one <i>Rps7</i>+/+ and two different <i>Rps7^Zma/+</i> 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 <i>Rps7</i>+/+ and two different <i>Rps7^Zma/+</i> embryos at E18.5 (G–I). The eye dysmorphology of <i>Rps7^Zma/+</i>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.</p

<i>Rps7^Mtu</i> shows reduced function in ribosomal precursor processing.

<p>(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 <i>Rps7^Mtu/+</i> (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 <i>Rps7</i>+/+ and <i>Rps7^Mtu/+</i> (* indicates p<0.01). The average of the values is reported in the bar graphs with S.E.M.</p