The miR‐379/miR‐410 cluster at the imprinted Dlk1‐Dio3 domain controls neonatal metabolic adaptation
2014; Springer Nature; Volume: 33; Issue: 19 Linguagem: Inglês
10.15252/embj.201387038
ISSN1460-2075
AutoresStéphane Labialle, Virginie Marty, Marie‐Line Bortolin‐Cavaillé, Magali Hoareau-Osman, Jean‐Philippe Pradère, Philippe Valet, Pascal G.P. Martin, Jérôme Cavaillé,
Tópico(s)RNA modifications and cancer
ResumoArticle14 August 2014free access Source Data The miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain controls neonatal metabolic adaptation Stéphane Labialle Stéphane Labialle Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Virginie Marty Virginie Marty Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Marie-Line Bortolin-Cavaillé Marie-Line Bortolin-Cavaillé Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Magali Hoareau-Osman Magali Hoareau-Osman Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Jean-Philippe Pradère Jean-Philippe Pradère Institut National de la Santé et de la Recherche Médicale (INSERM), U1048 Toulouse, France Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université de Toulouse, Université Paul Sabatier, Toulouse, France Search for more papers by this author Philippe Valet Philippe Valet Institut National de la Santé et de la Recherche Médicale (INSERM), U1048 Toulouse, France Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université de Toulouse, Université Paul Sabatier, Toulouse, France Search for more papers by this author Pascal GP Martin Pascal GP Martin Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France INRA, UMR1331, TOXALIM (Research Centre in Food Toxicology), Toulouse, France Université de Toulouse, INP, UPS, TOXALIM, Toulouse, France Search for more papers by this author Jérôme Cavaillé Corresponding Author Jérôme Cavaillé Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Stéphane Labialle Stéphane Labialle Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Virginie Marty Virginie Marty Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Marie-Line Bortolin-Cavaillé Marie-Line Bortolin-Cavaillé Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Magali Hoareau-Osman Magali Hoareau-Osman Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Jean-Philippe Pradère Jean-Philippe Pradère Institut National de la Santé et de la Recherche Médicale (INSERM), U1048 Toulouse, France Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université de Toulouse, Université Paul Sabatier, Toulouse, France Search for more papers by this author Philippe Valet Philippe Valet Institut National de la Santé et de la Recherche Médicale (INSERM), U1048 Toulouse, France Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université de Toulouse, Université Paul Sabatier, Toulouse, France Search for more papers by this author Pascal GP Martin Pascal GP Martin Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France INRA, UMR1331, TOXALIM (Research Centre in Food Toxicology), Toulouse, France Université de Toulouse, INP, UPS, TOXALIM, Toulouse, France Search for more papers by this author Jérôme Cavaillé Corresponding Author Jérôme Cavaillé Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France CNRS, LBME, UMR5099 Toulouse, France Search for more papers by this author Author Information Stéphane Labialle1,2,‡, Virginie Marty1,2,‡, Marie-Line Bortolin-Cavaillé1,2, Magali Hoareau-Osman1,2, Jean-Philippe Pradère3,4, Philippe Valet3,4, Pascal GP Martin1,2,5,6 and Jérôme Cavaillé 1,2 1Laboratoire de Biologie Moléculaire Eucaryote, UPS, Université de Toulouse, Toulouse, France 2CNRS, LBME, UMR5099 Toulouse, France 3Institut National de la Santé et de la Recherche Médicale (INSERM), U1048 Toulouse, France 4Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université de Toulouse, Université Paul Sabatier, Toulouse, France 5INRA, UMR1331, TOXALIM (Research Centre in Food Toxicology), Toulouse, France 6Université de Toulouse, INP, UPS, TOXALIM, Toulouse, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 561335934; Fax: +33 561335886; E-mail: [email protected] The EMBO Journal (2014)33:2216-2230https://doi.org/10.15252/embj.201387038 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In mammals, birth entails complex metabolic adjustments essential for neonatal survival. Using a mouse knockout model, we identify crucial biological roles for the miR-379/miR-410 cluster within the imprinted Dlk1-Dio3 region during this metabolic transition. The miR-379/miR-410 locus, also named C14MC in humans, is the largest known placental mammal-specific miRNA cluster, whose 39 miRNA genes are expressed only from the maternal allele. We found that heterozygote pups with a maternal—but not paternal—deletion of the miRNA cluster display partially penetrant neonatal lethality with defects in the maintenance of energy homeostasis. This maladaptive metabolic response is caused, at least in part, by profound changes in the activation of the neonatal hepatic gene expression program, pointing to as yet unidentified regulatory pathways that govern this crucial metabolic transition in the newborn's liver. Not only does our study highlight the physiological importance of miRNA genes that recently evolved in placental mammal lineages but it also unveils additional layers of RNA-mediated gene regulation at the Dlk1-Dio3 domain that impose parent-of-origin effects on metabolic control at birth and have likely contributed to mammal evolution. Synopsis Maternal expression of the imprinted miRNA cluster miR-379/miR-410 is central for metabolic adaptation in new-born mice, uncovering a physiological role for these miRNAs in regulating neonatal hepatic gene expression. Genetic ablation of the mouse imprinted miR-379/miR-410 cluster (C14MC in human) leads to neonatal lethality with incomplete penetrance. A subset of miR-379/miR-410-deficient pups have difficulties maintaining energy homeostasis in the safe range. Lack of miR-379/miR-410 expression is associated with profound changes in the neonatal hepatic gene expression program at the transition from fetal to post-natal life. This phenotype suggests a pivotal role for the miR-379/miR-410 cluster in neonatal survival very likely by controlling metabolic adaptation at birth. Introduction MicroRNAs (miRNAs) are endogenously expressed, ~19–23 nt-long non-coding RNAs (ncRNA) that silence gene expression at the post-transcriptional level, mostly via imperfect base-pairing interactions that occur preferentially within the 3′ untranslated regions (UTRs) of target mRNAs (Fabian & Sonenberg, 2012). Through their ability to target hundreds, perhaps even thousands of mRNAs, miRNAs are now considered as potent post-transcriptional regulators in an ever-growing list of developmental, physiological, or pathological contexts (Bushati & Cohen, 2007). Most conclusions drawn so far for mammalian miRNAs rely on computational mRNA target predictions coupled to gain- or loss-of-function approaches conducted mostly in vitro using cellular models. Although informative, these approaches do not undisputedly demonstrate the physiological significance of miRNAs. In that context, most miRNA knockout (KO) mice described so far do not exhibit overt abnormalities under classical mouse husbandry conditions, although defects can become apparent in physiologically challenging contexts (Leung & Sharp, 2010; Mendell & Olson, 2012). Accordingly, we still need to determine precisely, at the whole-organism level, the extent to which defects in miRNA-mediated regulation yield clear and interpretable phenotypic consequences (Park et al, 2010, 2012). In mammals, a subset of poorly conserved miRNA genes is subjected to genomic imprinting, a developmentally regulated form of epigenetic regulation that causes mono-allelic expression in a parent-of-origin dependent manner. That is, for a given gene, only one of the two parental alleles is transcriptionally competent (Barlow & Bartolomei, 2014). Most imprinted miRNA genes identified so far are organized as large (~40–100 kb) non-protein-coding transcriptional arrays that generate a large number (~50–100) of RNA species of related sequences. These clustered, tandemly repeated miRNAs likely arose through segmental duplication followed by sequence diversification and are presumably co-expressed as, and processed from, a single (or a few) long primary non-coding transcript(s). Remarkably, this unusual mode of genomic organization and epigenetic regulation appears specific to imprinted loci (Labialle & Cavaille, 2011) and has been reported at three evolutionarily distinct chromosomal regions: the eutherian-specific Dlk1-Dio3 domain (Seitz et al, 2003), the primate-specific C19MC region (Noguer-Dance et al, 2010), and the rodent-specific Sfmbt2 cluster (Wang et al, 2011). The ~1-Mbp eutherian-specific imprinted Dlk1-Dio3 chromosomal region on distal mouse chromosome 12 (orthologous to human chromosome 14q32) expresses three paternally expressed protein-coding genes (Dlk1, Rtl1, and Dio3) and several maternally expressed non-protein-coding transcripts, including Gtl2 as well as numerous box C/D small nucleolar (sno) RNAs and miRNAs (da Rocha et al, 2008) (Fig 1A). Many of these small RNAs are embedded within, and processed from, introns of poorly characterized long ncRNAs: In the mouse, many snoRNAs and miRNAs are intron-encoded within transcripts called Rian and Mirg, respectively. Most miRNA genes are grouped into two main regions, the miR-127/miR-136 cluster originating from an anti-Rtl1 transcript and the miR-379/miR-410 cluster (also referred to as C14MC in human). A few more are found scattered along the Dlk1-Dio3 region. The miR-127/miR-136 cluster silences the paternally expressed Rtl1 mRNAs through siRNA-like mechanisms (Seitz et al, 2003; Davis et al, 2005), and this trans-allelic RNAi-mediated regulation plays critical roles in placental development (Sekita et al, 2008). Although numerous functions for the miR-379/miR-410 cluster have been inferred from aberrant expression in pathological contexts or from enforced or knockdown expression experiments performed in vitro (Labialle & Cavaille, 2011; Benetatos et al, 2012; Girardot et al, 2012), the biological importance of this large miRNA cluster remains elusive. Of note, several studies have pointed out regulatory roles in neuronal functions, especially for miR-134 (Schratt et al, 2006; Fiore et al, 2009; Christensen et al, 2010; Gao et al, 2010; Jimenez-Mateos et al, 2012; Bicker et al, 2013; Rago et al, 2014). A correct dosage of imprinted genes encoded at the mouse Dlk1-Dio3 genomic interval is essential for embryonic growth and postnatal survival, as well as for muscle, skeletal, placental, and neuronal development (da Rocha et al, 2008). The functional significance of the corresponding imprinted human genes at 14q32 is supported by human syndromes of respiratory insufficiency, altered thoracic development, mental retardation, obesity, growth retardation, and precocious puberty (Kagami et al, 2008). The Dlk1-Dio3 genomic interval has also been linked genetically to diabetes (Wallace et al, 2010; Kameswaran et al, 2014). Whether the miR-379/miR-410 (C14MC) cluster contributes to one (or several) of these physiological processes or pathological contexts remains an open question. Figure 1. Targeted deletion of the miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain Schematic representation of the ˜1-Mbp imprinted Dlk1-Dio3 region on mouse distal chromosome 12. Paternally expressed protein-coding genes (Dlk1, Rtl1, and Dio3) are symbolized by blue rectangles, while maternally expressed miRNA and C/D snoRNA genes are depicted by pink stem loops and ovals, respectively. Gtl2 (pink rectangle) is a long, maternally expressed non-coding RNA (ncRNA) gene. Anti-Rtl1, Rian, and Mirg correspond to poorly characterized maternally expressed ncRNAs from which some, but not all, miRNAs and C/D snoRNAs are processed. It should be noted, however, that some deposited RNA sequences may simply represent RNA species whose functionality, if any, remains questionable (Chiang et al, 2010). Arrows indicate the sense of transcription, with the horizontal broken line highlighting the notion that Gtl2, anti-Rtl1, Rian, and Mirg may belong to the same transcription unit. Differentially methylated regions, including the imprinting center (Ig-DMR) that controls imprinted expression over the domain, are indicated by filled and open lollipops (methylated and un-methylated, respectively). The relative positions of hairpin-like (pre-miRNA) structures within the miR-379/miR-410 cluster are indicated in the enlarged inset. Note that most pre-miRNA genes at the 3′ end of the cluster are positioned within introns of Mirg (gray rectangles and dotted lines represent exons and splicing events, respectively). Top: The tissue-specific expression pattern of miRNAs was assayed by Northern blot analysis of adult mouse tissues as indicated on the panel, using a mixture of 32P-labelled oligonucleotides antisense to some miRNAs scattered along the cluster (miR-411, 323, 376b, 376a, 134, 154, and 410). The same membrane was probed with a let-7 oligo probe (gel loading control). Bottom: The tissue-specific expression pattern of Mirg host gene transcripts was assayed by RT–qPCR relative to Gapdh using the same set of tissues. Note that Mirg expression, but not that of miRNAs, is detected in testes, indicating that post-transcriptional regulation may occur in this tissue. WAT: white adipose tissue. Data are expressed as mean ± s.e.m. Cre/loxP-mediated site-specific deletion of the miR-379/miR-410 cluster. Left: Targeting strategy for disrupting the miR-379/miR-410 cluster through two independent homologous recombination events. Genome coordinates: UCSC Genome Browser, mouse, NCB137/mm9. Red and brown arrows indicate loxP and FRT recognition sites for the Cre and Flp site-specific recombinases, respectively. Right: PCR confirmation of the deletion using appropriate P1, P2, and P3 primers. The sequence of the deleted region was further confirmed by DNA sequencing. ΔMat and ΔPat represent heterozygous individuals with maternally and paternally inherited deletions, respectively, while WT correspond to wild-type littermate controls. M: DNA ladder (bp). Source data are available online for this figure. Source Data for Figure 1 [embj201387038-SourceData-Fig1.pdf] Download figure Download PowerPoint Newly available epigenetically regulated arrays of miRNA genes raise intriguing questions regarding the evolutionarily meaning of their repeated structures in relation to their mono-allelic expression patterns and the potential redundancy or divergence in the functions of related miRNA genes within a given cluster (Labialle et al, 2011). Studying these imprinted arrays of miRNA genes therefore provides unique opportunities to address the physiological roles of lineage-specific miRNAs, but also to evaluate how a large number of coordinately expressed miRNAs can impact gene regulatory networks. To elucidate the biological roles of the large miR-379/miR-410 cluster at the Dlk1-Dio3 domain, we have used a constitutive mouse KO model. Unexpectedly, a subset of miR-379/miR-410-deficient neonates failed to maintain energy homeostasis and die shortly after birth. These neonatal metabolic deficiencies—affecting both lipid and glucose metabolism—are very likely caused, at least in part, by defects in the temporal activation of a large set of metabolic genes in the newborn's liver. This is, to the best of our knowledge, the first demonstration that miRNAs exert critical functions at the transition from fetal to postnatal life in mammals. Results Targeted disruption of the miR-379/miR-410 cluster The imprinted Dlk1-Dio3 domain contains the largest eutherian-specific miRNA cluster: The miR-379/miR-410 cluster (Fig 1A), named C14MC in humans, believed to produce 77 and 63 mature miRNAs in the mouse and humans, respectively (http://www.mirbase.org/). In the adult mouse, miR-379/miR-410 expression is mostly restricted to the brain (Fig 1B). In contrast, during development and also at birth, these miRNAs are widely expressed in many non-brain tissues (Fig 2A–C, and Supplementary Fig S4). To assess the biological functions of miR-379/miR-410 in vivo, we created a constitutive KO mouse model carrying a Cre/loxP-mediated ~59-kb-long deletion (Fig 1C) that removes the entire miRNA cluster (denoted as ΔmiR). Figure 2. Targeted deletion of the miR-379/miR-410 cluster does not affect the expression levels of flanking genes Expression of the miR-379/miR-410 cluster and its flanking C/D snoRNA (MBII-78, MBII-48) and miRNA (miR-127) genes was assayed by Northern blot analysis of the indicated samples (two individuals per genotype). Expression of the miR-379/miR-410 cluster was assayed by Northern blot analysis of the dissected tissues indicated above the panels from WT or ΔMat E18.5 embryos. A tRNA-specific probe was used as the internal loading control. Expression of selected miRNAs (miR-379, miR-376a, and miR-410) was assayed by RT–qPCR relative to U6 snRNA in P0 tissues prepared from WT or ΔMat individuals (n = 3 per genotype), as indicated below the histograms. BAT: brown adipose tissue. Data are expressed as mean ± s.e.m. Expression of mRNAs (Dlk1 and Dio3) and mRNA-like transcripts (Gtl2, Rian, Mirg) was assayed by RT–qPCR relative to Gapdh mRNA in the indicated tissues. Blue and pink bars represent expression levels in ΔPat and ΔMat individuals, respectively (six individuals per genotype). Expression levels of WT were arbitrarily set to 1. Source data are available online for this figure. Source Data for Figure 2A, B [embj201387038-SourceData-Fig2A-B.pdf] Download figure Download PowerPoint The miR-379/miR-410 cluster is regulated by genomic imprinting; only the maternally inherited allele is competent for transcription (Seitz et al, 2003). Accordingly, we analyzed ΔmiR mice generated by two types of crosses: (i) wild-type females with heterozygous males (paternal inheritance of the targeted deletion) and (ii) wild-type males with heterozygous females (maternal inheritance of the targeted deletion). These two reciprocal crosses are expected to generate 50% wild-type and 50% heterozygous animals. In principle, only heterozygotes having inherited the deletion from their mother (denoted as ΔMat) are deficient for miR-379/miR-410 expression. In contrast, heterozygotes having inherited the deletion from their father (denoted as ΔPat) express the miRNA cluster normally. We therefore analyzed ΔMat and ΔPat animals since these two classes of heterozygotes are genetically comparable, thus alleviating any potential confounding effects due to differences in genetic background. Wild-type littermates (denoted as WT) were also used as controls. We first validated our KO model by demonstrating that, as expected, upon maternal but not paternal inheritance, the targeted miRNA genes were no longer expressed in embryo (E18.5), placenta (E18.5), and adult brain (Fig 2A). More sensitive RT–qPCR experiments revealed very weak, but still detectable, expression for some individual miRNAs (Fig 2C) or for the Mirg host gene in the embryo (Fig 2D), indicating that leaky expression (~5%) can occur on the normally silenced paternal allele in some tissues. More importantly, the level of expression of the surrounding imprinted ncRNA and protein-coding genes (Fig 2D), as well as their imprinted status (Supplementary Fig S1), remained unaffected, making it very unlikely that ΔmiR removes important regulatory elements that govern imprinted expression over the Dlk1-Dio3 genomic interval. Genetic ablation of the miR-379/miR-410 cluster leads to partially penetrant neonatal lethality After extensive breeding in the C57BL/6J genetic background, we found that, upon maternal but not paternal inheritance, ~39% of ΔMat animals were missing at 3–4 weeks of age (Table 1). This alteration in the Mendelian ratio was likewise observed in crosses where the dams inherited the deletion paternally or maternally, thus excluding any grand-parental effects (Table 2). ΔmiR does not lead to embryonic lethality since ΔMat concepti reached late gestation (E18.5) at the expected Mendelian frequencies (Table 1) without any obvious alteration in overall growth of the embryos or placenta (Supplementary Fig S2). ΔMat pups were born alive, but half of them died within ~15–40 h post-delivery (Table 3). Maternal care was normal as were the weights and overall appearance of major neonatal organs we examined (Supplementary Fig S2). Once neonates overcame the fetal-to-postnatal transition, survivors did not exhibit any significant increase in lethality over at least 8 months. However, upon maternal, but not paternal, inheritance, both males and females were transiently growth-retarded ~3 weeks after birth, coinciding with the suckling–weaning transition (Supplementary Fig S2). Table 1. Maternally, but not paternally inherited ΔmiR, leads to altered Mendelian ratios 4 weeks after birth but not before birth (E18.5) C57BL/6J (N6–N12) 4-week-old mice E18.5 embryos Maternal inheritance Paternal inheritance Maternal inheritance Paternal inheritance Total individuals 753 656 65 54 Litter (n) 148 108 11 8 Sex Males 378 306 n.d. n.d. Females 375 350 n.d n.d. Observed genotypes WT 468 346 33 27 Heterozygotes 285 310 32 27 P-values 2.57e−11 0.16 0.9 1.0 Table 2. The altered Mendelian ratio 4 weeks after birth is not caused by the lack of expression of the miR-379/miR-410 cluster in the dams Parental origin of ΔmiR in the dams Paternal (miRNA expression) Maternal (no miRNA expression) Total individuals 472 281 Litter (n) 92 56 Sex Males 247 131 Females 225 150 Observed genotypes WT 298 170 ΔMat 174 111 P-values 1.14e−8 4.32e−4 Table 3. ΔMat neonates are born alive but die within 15–40 h after birth Neonates (P0–P3) Maternal inheritance Total individuals 93 Litter (n) 14 Time after birth 0–14 h 15–40 h 41–96 h Observed genotypes WT 39 (6) 36 (3) 36 (0) ΔMat 43 (5) 22 (21) 20 (2) P-values 0.76 0.0002 n.d. Numbers in parentheses represent dead pups, and P-values estimate the significance of the unequal number of dead pups in each genotype by the χ2 test. Genetic ablation of the miR-379/miR-410 cluster results in impaired neonatal glucose homeostasis Neonatal death can result from a large spectrum of physiological defects (Turgeon & Meloche, 2009). In the absence of any apparent morphological defects and due to the timing of lethality, we hypothesized that ΔMat neonates may have deficiencies in maintaining energy homeostasis. Indeed, birth entails major metabolic challenges as the energy supply provided mostly as glucose by placental nutrition suddenly ceases. To combat this naturally occurring starvation episode, neonates first degrade their hepatic glycogen store constituted during late embryogenesis (glycogenolysis) and then activate gluconeogenesis, which is not active in the immediate postnatal period (Girard et al, 1992). As shown in Fig 3A, blood glucose levels at P0 were reduced by ~20% in ΔMat neonates relative to WT littermates while, as expected, blood sugar concentration in ΔPat neonates was in the normal range. Interestingly, the distribution of glycemic status at P1 revealed that a subset of ΔMat neonates displayed severe hypoglycemia (defined here arbitrarily as glucose levels < 30 mg/dl) while glucose levels in the remaining mutants were only slightly lower than those of WT (Fig 3A and Supplementary Fig S3). A careful tracking showed that mutant dead pups collected between P0 and P1 together with profoundly hypoglycemic individuals at P1 account for 37.7% of ΔMat individuals (n = 17 litters, Table 4). Given the neonatal death observed around birth (Table 3) and the deficit of ΔMat individuals at weaning (Table 1), most of these severely hypoglycemic mutants are unlikely to survive the perinatal period, thus implying that hypoglycemia contributes to neonatal death. To challenge this assumption, WT and ΔMat neonates were systematically given subcutaneous glucose injections (50 μl, 10%) shortly after birth and then 4–6 h later (n = 10 litters). In order to avoid any bias due to the incomplete penetrance of the lethality, we only considered litters without any dead pups at the time of the first injections. As reported in Fig 3G, glucose injections rescued, at least in part, the neonatal lethality phenotypes since 25/31 (80.6%) glucose-injected mutants survived birth transition, with 24/25 still alive after weaning. In comparison, the perinatal survival rate of ΔMat without glucose injections was 42% (Table 3). We next sought to further appreciate the contribution of deficiencies in glucose homeostasis by studying pups kept separated from their mother and maintained in a moist, warm (32–35°C) atmosphere. This procedure alleviates confounding variables in that external sources of glucose cannot be provided by mother's milk. In a first pilot experiment during which we monitored continuously the fate of neonates, no ΔMat (0/10) neonates survived starvation over a 10-h period while 60% of WT (11/18) were still alive over the same time course (n = 4 litters; Fig 3H, left). In a second set of experiments, we asked whether glucose injections may extend the lifespan of ΔMat pups in such challenging metabolic environment. For each litter, half of the pups were injected subcutaneously with glucose, immediately after birth and then every 6 h over a period of 12 h. The remaining 50% of pups were injected with NaCl (50 μl, 0.9%) and used as controls (n = 5 litters). As shown in Fig 3H (right), lethality of ΔMat neonates could be delayed since 7/7 (100%) mutants and 5/7 (71%) WT pups injected with glucose survived up to 17 h. In comparison, all NaCl-injected control individuals were dead 17 h post-injection. The survival of 6/7 NaCl-injected ΔMat neonates at t + 10 h reflects probably the notion that hydration of pups contributes to their survival when no access to mother's milk is allowed. Altogether, these glucose rescue experiments demonstrate that dysregulation of glycemia is a major early event linked to the neonatal lethality. Figure 3. Maternal, but not paternal, deletion of the miR-379/miR-410 cluster impairs hepatic glycogenolysis and gluconeogenesis A–F. Serum glucose (A, B), hepatic glycogen (C, D), insulin (E), glucagon (F) levels in WT (black), ΔMat (pink), severely hypoglycemic ΔMat (dark pink) or ΔPat (blue) neonates were measured in vaginally delivered neonates (P0, P1) or after cesarean delivery (E19.5, E19.5 + 1, E19.5 + 4). Insulin and glucagon levels of ΔMat individuals in the same cohort with the highest and lowest glucose concentration, denoted as high (50.78 ± 1.42 mg/dl) and low (27.38 ± 3.72 mg/dl), respectively, are also shown in the histograms to the right (E and F). Numbers of individuals analyzed are indicated above the histograms. G. Glucose injections rescue the neonatal lethality phenotype. Dotted lines represent the % of ΔMat neonatal survival as observed from dead pups collected perinatally (Table 3). H. Left: The lifespan of ΔMat neonates (pink line) is reduced relative to WT (black line) when P0 neonates were kept separated from their mother. Right: Glucose injections extend the lifespan of ΔMat neonates. Histograms show the survival rate of NaCl- or glucose-injected pups at 10 and 17 h post-injection. I. The temporal expression of the gluconeogenic Pck1 and G6pc genes in liver (relative to Gapdh) was assayed by RT–qPCR (6 individuals per genotype). Data information: Data are expressed as mean ± s.e.m. Download figure Download PowerPoint Table 4. The neonatal fate of WT and ΔMat pups between P0 and P1 Maternal inheritance (n = 17 litters) ΔMat (n = 69) WT (n = 61) Dead pups (P0–P1) 17 6 Hypoglycemic pups (P1) (glycemic status < 30 mg/dl) 9 2 Dead + hypoglycemic pups (%) 37.7 13.1 Genetic ablation of miR-379/miR-410 cluster alters neonatal glycogenolysis and gluconeogenesis Because mobilization of glycogen stores is one of the very first metabolic adaptive events, we assessed hepatic glycogen levels. As shown in Fig 3C, ΔMat neonates at P0 had 40–50% higher hepatic glycogen contents than their WT littermates whereas, as expected, hepatic glycogen stores were in the normal range in ΔPat neona
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