Notchless affects the Wnt pathway during pre-implantation development in mice

In order to determine the genetic pathways required for early mammalian fetal development, a positional cloning study was performed using two embryonic lethal lines of mice, l11Jus1, and l11Jus4. These lines were derived from an ENU mutagenesis screen targeted to a 34Mb region of mouse chromosome 11. The mutations were identified for both lines in Notchless (Nle1), a regulator of the Notch pathway, and a gene expression study was initiated. RNA was isolated from wild type and l11Jus1 homozygous embryos for qRT- PCR analysis using a Notch pathway focused PCR array. Genes present on the array include Notch ligands, receptors, target genes, transcription factors, and genes from other related genetic pathways. Very few Notch target genes were misregulated in the mutants, while there was a non-significant down regulation of some of the ligands and receptors. Only Cdkn1a was significantly up-regulated in the mutants. Cdkn1a is a target gene of Notch signaling, but is also involved in apoptosis. Surprisingly, this study revealed a down-regulation of Wnt genes in the mutant embryos during pre-implantation development. Wnt and Notch are known to interact later in development during somitogenesis, but Notch is not required during pre-implantation development. This study shows a component of Notch signaling (i.e. Nle1) interacts with Wnt during peri-implantation in the developing embryo without affecting global Notch signaling. The over-expression of Cdkn1a could result from misregulation of Notch, disruption in another Nle1 dependent pathway during this developmental timepoint or because the embryo is undergoing apoptosis. Therefore, either Notch signaling interacts with Wnt during pre-implantation development, or Nle1 acts through a yet to be identified pathway that alters Wnt signaling

Deletion of \u3ci\u3eShank1\u3c/i\u3e Has Minimal Effects on the Molecular Composition and Function of Glutamatergic Afferent Postsynapses in the Mouse Inner Ear

Abstract Shank proteins (1-3) are considered the master organizers of glutamatergic postsynaptic densities in the central nervous system, and the genetic deletion of either Shank1, 2, or 3 results in altered composition, form, and strength of glutamatergic postsynapses. To investigate the contribution of Shank proteins to glutamatergic afferent synapses of the inner ear and especially cochlea, we used immunofluorescence and quantitative real time PCR to determine the expression of Shank1, 2, and 3 in the cochlea. Because we found evidence for expression of Shank1 but not 2 and 3, we investigated the morphology, composition, and function of afferent postsynaptic densities from defined tonotopic regions in the cochlea of Shank1(-/-) mice. Using immunofluorescence, we identified subtle changes in the morphology and composition (but not number and localization) of cochlear afferent postsynaptic densities at the lower frequency region (8 kHz) in Shank1(-/-) mice compared to Shank1(+/+) littermates. However, we detected no differences in auditory brainstem responses at matching or higher frequencies. We also identified Shank1 in the vestibular afferent postsynaptic densities, but detected no differences in vestibular sensory evoked potentials in Shank1(-/-) mice compared to Shank1(+/+) littermates. This work suggests that Shank proteins play a different role in the development and maintenance of glutamatergic afferent synapses in the inner ear compared to the central nervous system

ENU mutagenesis reveals that <it>Notchless homolog 1</it> (<it>Drosophila</it>) affects <it>Cdkn1a</it> and several members of the <it>Wnt</it> pathway during murine pre-implantation development

<p>Abstract</p> <p>Background</p> <p>Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Mutation analysis targeted to a 34 Mb domain flanked by <it>Trp53</it> and <it>Wnt3</it> demonstrates that this region of mouse chromosome 11 contains a large number of essential genes. Two mutant alleles (<it>l11Jus1</it> and <it>l11Jus4</it>), which fall into the same complementation group, survive through implantation but fail prior to gastrulation.</p> <p>Results</p> <p>Through a positional cloning strategy, we discovered that these homozygous mutant alleles contain non-conservative missense mutations in the <it>Notchless homolog 1</it> (<it>Drosophila</it>) (<it>Nle1</it>) gene. NLE1 is a member of the large WD40-repeat protein family, and is thought to signal via the canonical NOTCH pathway in vertebrates. However, the phenotype of the <it>Nle1</it> mutant mice is much more severe than single <it>Notch</it> receptor mutations or even in animals in which NOTCH signaling is blocked. To test the hypothesis that NLE1 functions in multiple signaling pathways during pre-implantation development, we examined expression of multiple <it>Notch</it> downstream target genes, as well as select members of the <it>Wnt</it> pathway in wild-type and mutant embryos. We did not detect altered expression of any primary members of the <it>Notch</it> pathway or in <it>Notch</it> downstream target genes. However, our data reveal that <it>Cdkn1a</it>, a NOTCH target, was upregulated in <it>Nle1</it> mutants, while several members of the <it>Wnt</it> pathway are downregulated. In addition, we found that <it>Nle1</it> mutant embryos undergo caspase-mediated apoptosis as hatched blastocysts, but not as morulae or blastocysts.</p> <p>Conclusions</p> <p>Taken together, these results uncover potential novel functions for NLE1 in the WNT and CDKN1A pathways during embryonic development in mammals.</p

Deletion of Shank1 has minimal effects on the molecular composition and function of glutamatergic afferent postsynapses in the mouse inner ear

Shank proteins (1-3) are considered the master organizers of glutamatergic postsynaptic densities in the central nervous system, and the genetic deletion of either Shank1, 2, or 3 results in altered composition, form, and strength of glutamatergic postsynapses. To investigate the contribution of Shank proteins to glutamatergic afferent synapses of the inner ear and especially cochlea, we used immunofluorescence and quantitative real time PCR to determine the expression of Shank1, 2, and 3 in the cochlea. Because we found evidence for expression of Shank1 but not 2 and 3, we investigated the morphology, composition, and function of afferent postsynaptic densities from defined tonotopic regions in the cochlea of Shank1(-/-) mice. Using immunofluorescence, we identified subtle changes in the morphology and composition (but not number and localization) of cochlear afferent postsynaptic densities at the lower frequency region (8 kHz) in Shank1(-/-) mice compared to Shank1(+/+) littermates. However, we detected no differences in auditory brainstem responses at matching or higher frequencies. We also identified Shank1 in the vestibular afferent postsynaptic densities, but detected no differences in vestibular sensory evoked potentials in Shank1(-/-) mice compared to Shank1(+/+) littermates. This work suggests that Shank proteins play a different role in the development and maintenance of glutamatergic afferent synapses in the inner ear compared to the central nervous system. (C) 2015 Elsevier B.V. All rights reserved

Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

Background The Global Burden of Diseases, Injuries, and Risk Factors Study 2016 (GBD 2016) provides a comprehensive assessment of risk factor exposure and attributable burden of disease. By providing estimates over a long time series, this study can monitor risk exposure trends critical to health surveillance and inform policy debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of GBD to estimate levels and trends in exposure, attributable deaths, and attributable disability-adjusted life-years (DALYs), by age group, sex, year, and location for 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2016. This study included 481 risk-outcome pairs that met the GBD study criteria for convincing or probable evidence of causation. We extracted relative risk (RR) and exposure estimates from 22 717 randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources, according to the GBD 2016 source counting methods. Using the counterfactual scenario of theoretical minimum risk exposure level (TMREL), we estimated the portion of deaths and DALYs that could be attributed to a given risk. Finally, we explored four drivers of trends in attributable burden: population growth, population ageing, trends in risk exposure, and all other factors combined. Findings Since 1990, exposure increased significantly for 30 risks, did not change significantly for four risks, and decreased significantly for 31 risks. Among risks that are leading causes of burden of disease, child growth failure and household air pollution showed the most significant declines, while metabolic risks, such as body-mass index and high fasting plasma glucose, showed significant increases. In 2016, at Level 3 of the hierarchy, the three leading risk factors in terms of attributable DALYs at the global level for men were smoking (124·1 million DALYs [95% UI 111·2 million to 137·0 million]), high systolic blood pressure (122·2 million DALYs [110·3 million to 133·3 million], and low birthweight and short gestation (83·0 million DALYs [78·3 million to 87·7 million]), and for women, were high systolic blood pressure (89·9 million DALYs [80·9 million to 98·2 million]), high body-mass index (64·8 million DALYs [44·4 million to 87·6 million]), and high fasting plasma glucose (63·8 million DALYs [53·2 million to 76·3 million]). In 2016 in 113 countries, the leading risk factor in terms of attributable DALYs was a metabolic risk factor. Smoking remained among the leading five risk factors for DALYs for 109 countries, while low birthweight and short gestation was the leading risk factor for DALYs in 38 countries, particularly in sub-Saharan Africa and South Asia. In terms of important drivers of change in trends of burden attributable to risk factors, between 2006 and 2016 exposure to risks explains an 9·3% (6·9–11·6) decline in deaths and a 10·8% (8·3–13·1) decrease in DALYs at the global level, while population ageing accounts for 14·9% (12·7–17·5) of deaths and 6·2% (3·9–8·7) of DALYs, and population growth for 12·4% (10·1–14·9) of deaths and 12·4% (10·1–14·9) of DALYs. The largest contribution of trends in risk exposure to disease burden is seen between ages 1 year and 4 years, where a decline of 27·3% (24·9–29·7) of the change in DALYs between 2006 and 2016 can be attributed to declines in exposure to risks. Interpretation Increasingly detailed understanding of the trends in risk exposure and the RRs for each risk-outcome pair provide insights into both the magnitude of health loss attributable to risks and how modification of risk exposure has contributed to health trends. Metabolic risks warrant particular policy attention, due to their large contribution to global disease burden, increasing trends, and variable patterns across countries at the same level of development. GBD 2016 findings show that, while it has huge potential to improve health, risk modification has played a relatively small part in the past decade. Funding The Bill & Melinda Gates Foundation, Bloomberg Philanthropies

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present