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UK Dementia Research Institute at The University of Cambridge, Cambridge, United KingdomDepartment of Clinical Neurosciences, School of Clinical Medicine, University of Cambridge, Cambridge, United Kingdom
). Rpl24 encodes the protein RPL24, which is incorporated into the ribosome in the cytoplasm. Notably, altered expression of five other ribosomal protein genes—Rps7, Rps21, Rps19, Rps20, and RACK1—result in belly spots (
). Despite this genetic evidence of a link between ribosomal proteins and belly spots, the mechanism(s) behind the phenotype are unknown. The Rpl24Bst mutation reduces protein synthesis in embryonic neural tube cells (
). Suppression of protein synthesis in Rpl24Bst variants is completely reversed by eEF2K inactivation, providing a tool to reverse the protein synthesis defects in Rpl24Bst variants. The Eef2kD273A allele is a germline knockin that dramatically reduces eEF2K catalytic activity throughout the whole mouse from conception (
). We therefore used the Eef2kD273A allele to assess the influence of eEF2K inactivation on Rpl24Bst phenotypes. To achieve this, belly spot and tail severity were scored using a scale from 0 (normal) to 4 (severe) for mice generated in our previous study (Supplementary Figure S1a). The combined belly-spot and tail score of Rpl24Bst/+ mice has a median of 3 of 8 (Figure 1a). In comparison, the belly-spot and tail score of Rpl24Bst/+Eef2kD273A/D273A mice was significantly lower at only 1.5. Furthermore, there is a lower incidence of severe phenotypes, with only 7% of Rpl24Bst/+Eef2kD273A/D273A mice scoring ≥5 compared with 34% of Rpl24Bst/+ mice. Therefore, inactivation of eEF2K suppresses the observable skin and tail phenotypes of the Rpl24Bst mutation. The main contributing factor to this difference is the tail score, with 35% of tails scoring >2 in Rpl24Bst/+ mice compared with 3.6% in Rpl24Bst/+Eef2kD273A/D273A mice (Supplementary Figure S1b).
Next, we took a complementary approach measuring the effect of the Rpl24Bst and Eef2kD273A mutations on melanocytes using the Dct-lacZ system (
). Genetically engineered Dct-lacZ mice express β-galactosidase from the melanocyte-specific dopachrome tautomerase (Dct) promoter, allowing whole-mount visualization and quantification of melanocyte location. On embryonic day 13.5, melanocytes are transiting the forelimbs of embryos, with the fraction of melanocyte-positive forelimb a metric for changes in melanocyte migration. We observe a significant reduction in melanocyte migration in Rpl24Bst/+ embryos, consistent with their belly spots in adulthood, whereas the inactivation of eEF2K has no effect (Figure 1b). Compared with that in Rpl24Bst/+ embryos, melanocyte migration is significantly reverted in Rpl24Bst/+Eef2kD273A/D273A embryos (Figure 1b). Thus, eEF2K activity is required for the perturbed melanocyte migration phenotype of Rpl24Bst embryos. Why the reverted migration in Rpl24Bst/+Eef2kD273A/D273A embryos (Figure 1b) does not correlate with a reversal of adult belly spot phenotype (Supplementary Figure S1b) is unclear. It is possible that migration is reversed at embryonic day 13.5 but subsequently slows to produce a belly spot. Unfortunately, staining of Dct-lacZ is not possible at later embryonic stages owing to reduced skin permeabilization (
We noted that Rpl24Bst/+ and Rpl24Bst/+Eef2kD273A/D273A mice were weaned at lower than Mendelian frequencies: Rpl24Bst/+ mice at 1 in 3 (32.1%) when expected at 1 in 2 and Rpl24Bst/+Eef2kD273A/D273A mice even less frequently at 1 in 5 (20.7%) (Figure 2a). Unexpectedly, when we compared the frequency of Rpl24Bst/+Eef2kD273A/D273A mice with the experimentally determined frequency of Rpl24Bst/+ mice (32.1%), we found this to be significantly different (Figure 2a). Thus, the viability of Rpl24Bst/+ mice is at least in part dependent on eEF2K activity. To analyze this effect further, we calculated the frequencies of the same genotypes in embryonic day 13.5 embryos, finding that the incidence of the Rpl24Bst mutation was close to Mendelian with active or inactive eEF2K (Figure 2b). From this, we conclude that the reduced viability of Rpl24Bst/+Eef2kD273A/D273A mice occurs between embryonic day 13.5 and weaning. Therefore, despite being responsible for the skin and tail phenotypes of the Rpl24Bst mutation, eEF2K promotes the preweaning survival of Rpl24Bst-variant mice. Embryo viability could not be determined during our analysis. Thus, Rpl24Bst/+Eef2kD273A/D273A embryos with the greatest reversion in belly-spot phenotype may not survive, potentially explaining the observed discrepancy in melanocyte migration and adult belly spots in this genotype. These data provide substantive evidence of a mechanistic link between RPL24 and eEF2K at an organism level.
Similar to eEF2K inactivation, deletion of p53 in Rpl24Bst variants reversed belly-spot and tail defects while also reducing the ratio of Rpl24Bst/+ mice (
). Rpl24Bst/+p53‒/‒ mice showed reduced embryonic apoptosis and dependence on p21 for viability. Suggesting a shared mechanism, eEF2K activity is required for apoptosis at various stages of development and promotes p21 expression (
). Whether inactivation of eEF2K would also rescue these belly-spot phenotypes should now be determined
This work extends the relationship between RPL24 and eEF2K to include melanocyte migration, tail deformation, and organism survival. Rpl24Bst/+ mice have been used in cancer studies, to model retinal degeneration, and to study brain development (
). This work shows that eEF2K may play a role across these diverse biological settings, with further work merited to investigate its importance.
Declaration for animal use
Studies were carried out under license from the UK Home Office (60/4183 and 70/8646). Mice were maintained in open-top cages with a 12-hour light/dark cycle and free access to water and diet. Experiments were initiated on inbred C57BL/6J male and female mice aged between 6 and 12 weeks, without blinding or randomization.
Data availability statement
No datasets were generated or analyzed during this study.
Funding was provided by Cancer Research UK ( A17196 , A24388 , A21139 , A31287 ) and H2020 European Research Council (311301) to OJS; by the Wellcome Trust (201487) to GRM, TvdH, CMS, OJS, and AEW; and by the National Health and Medical Research Council to CGP.
Supplementary Materials and Methods
All materials are freely available, where legally permitted, on acceptance of a material transfer agreement.
Belly-spot and tail scoring
Genotyping was carried out by Transnetyx (Memphis, TN). Scoring was carried out at the endpoint of published experiments that determined sample size, with no power analysis performed for this study. Scoring was performed regardless of the presence of other alleles such as VillinCreER, Apcfl, ApcMin, or KrasG12D because these alleles do not manifest a belly-spot or tail phenotype.
Overnight vaginal plugs were designated embryonic day 0.5, and embryos were collected on embryonic day 13.5. Embryo studies were performed blind. Tails were used for genotyping, and the remainder were fixed in ice-cold 0.25% glutaraldehyde (G6257, Sigma-Aldrich, St. Louis, MO) in PBS, on a rotational mixer for 30 minutes at 4 °C. Embryos were washed in cold PBS and permeabilized in two rounds of 2 mM magnesium chloride, 0.1% sodium deoxycholate (D6750, Sigma-Aldrich), and 0.02% NP40 (74385, Sigma-Aldrich) in PBS for a total of 40 minutes at room temperature. Embryos were stained in 2 mM magnesium chloride, 0.1% sodium deoxycholate, 0.02% NP40, 4.7 mM potassium hexacyanoferrate (II) trihydrate (P9387, Sigma-Aldrich), 4.8 mM potassium hexacyanoferrate (III) (P8131, Sigma-Aldrich), and 0.5 mg/ml X-Gal (V394A, Promega, Madison, WI) in PBS at 4 °C on a rotational mixer protected from light for 36 hours. Embryos were washed in PBS, visualized by light microscopy, and stored in formalin. The sample size required for significance was estimated on the basis of pilot studies using G∗Power (