Alternative splicing is frequent during early embryonic development in mouse

<p>Abstract</p> <p>Background</p> <p>Alternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development.</p> <p>Results</p> <p>A genome-wide analysis of alternative isoform expression in embryonic day 8.5, 9.5 and 11.5 mouse embryos and placenta was carried out using a splicing-sensitive exon microarray. We show that alternative splicing and isoform expression is frequent across developmental stages and tissues, and is comparable in frequency to the variation in whole-transcript expression. The genes that are alternatively spliced across our samples are disproportionately involved in important developmental processes. Finally, we find that a number of RNA binding proteins, including putative splicing factors, are differentially expressed and spliced across our samples suggesting that such proteins may be involved in regulating tissue and temporal variation in isoform expression. Using an example of a well characterized splicing factor, <it>Fox2</it>, we demonstrate that changes in <it>Fox2 </it>expression levels can be used to predict changes in inclusion levels of alternative exons that are flanked by Fox2 binding sites.</p> <p>Conclusions</p> <p>We propose that alternative splicing is an important developmental regulatory mechanism. We further propose that gene expression should routinely be monitored at both the whole transcript and the isoform level in developmental studies</p

Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation

International audienceAnterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses

Craniofacial Defects in Embryos with Homozygous Deletion of Eftud2 in Their Neural Crest Cells Are Not Rescued by Trp53 Deletion

Embryos with homozygous mutation of Eftud2 in their neural crest cells (Eftud2ncc&minus;/&minus;) have brain and craniofacial malformations, hyperactivation of the P53-pathway and die before birth. Treatment of Eftud2ncc&minus;/&minus; embryos with pifithrin-&alpha;, a P53-inhibitor, partly improved brain and craniofacial development. To uncover if craniofacial malformations and death were indeed due to P53 hyperactivation we generated embryos with homozygous loss of function mutations in both Eftud2 and Trp53 in the neural crest cells. We evaluated the molecular mechanism underlying craniofacial development in pifithrin-&alpha;-treated embryos and in Eftud2; Trp53 double homozygous (Eftud2ncc&minus;/&minus;; Trp53ncc&minus;/&minus;) mutant embryos. Eftud2ncc&minus;/&minus; embryos that were treated with pifithrin-&alpha; or homozygous mutant for Trp53 in their neural crest cells showed reduced apoptosis in their neural tube and reduced P53-target activity. Furthermore, although the number of SOX10 positive cranial neural crest cells was increased in embryonic day (E) 9.0 Eftud2ncc&minus;/&minus;; Trp53ncc&minus;/&minus; embryos compared to Eftud2ncc&minus;/&minus; mutants, brain and craniofacial development, and survival were not improved in double mutant embryos. Furthermore, mis-splicing of both P53-regulated transcripts, Mdm2 and Foxm1, and a P53-independent transcript, Synj2bp, was increased in the head of Eftud2ncc&minus;/&minus;; Trp53ncc&minus;/&minus; embryos. While levels of Zmat3, a P53- regulated splicing factor, was similar to those of wild-type. Altogether, our data indicate that both P53-regulated and P53-independent pathways contribute to craniofacial malformations and death of Eftud2ncc&minus;/&minus; embryos

Loss of function mutation of Eftud2, the gene responsible for mandibulofacial dysostosis with microcephaly (MFDM), leads to pre-implantation arrest in mouse.

Mutations in EFTUD2 are responsible for the autosomal dominant syndrome named MFDM (mandibulofacial dysostosis with microcephaly). However, it is not clear how reduced levels of EFTUD2 cause abnormalities associated with this syndrome. To determine if the mouse can serve as a model for uncovering the etiology of abnormalities found in MFDM patients, we used in situ hybridization to characterize expression of Eftud2 during mouse development, and used CRISPR/Cas9 to generate a mutant mouse line with deletion of exon 2 of the mouse gene. We found that Eftud2 was expressed throughout embryonic development, though its expression was enriched in the developing head and craniofacial regions. Additionally, Eftud2 heterozygous mutant embryos had reduced EFTUD2 mRNA and protein levels. Moreover, Eftud2 heterozygous embryos were born at the expected Mendelian frequency, and were viable and fertile despite being developmentally delayed. In contrast, Eftud2 homozygous mutant embryos were not found post-implantation but were present at the expected Mendelian frequency at embryonic day (E) 3.5. Furthermore, only wild-type and heterozygous E3.5 embryos survived ex vivo culture. Our data indicate that Eftud2 expression is enriched in the precusor of structures affected in MFDM patients and show that heterozygous mice carrying deletion of exon 2 do not model MFDM. In addition, we uncovered a requirement for normal levels of Eftud2 for survival of pre-implantation zygotes

Increased NAFLD in <i>Tmed2</i>^<i>99J/+</i> mice.

<p>Representative images of Hematoxylin & Eosin stained liver sections showing A). a healthy liver section; and phenotypes scored for on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182995#pone.0182995.t001" target="_blank">Table 1</a>; B). macrosteatosis; C). microsteatosis D). lobular inflammation; E). portal inflammation; and F). ballooning. G. Significantly more <i>Tmed2</i>^<i>99J/+</i>mice had NAFLD scores of ≥ 4 when compared to age-matched wildtype controls. **P<0.01 using Fisher exact t-test. Arrows indicate inflammatory cells. Scale bar = 50um. WT = Wildtype.</p

Expression of lipid biosynthesis regulators-SREBPs in wildtype and <i>Tmed2</i>^<i>99J/+</i> livers.

<p>A). <i>Srebp1α</i> level was increased in <i>Tmed2</i>^<i>99J/+</i> increased in P5 <i>Tmed2</i>^<i>99J/+</i> mice compared to age-matched wildtype controls. B). <i>Srebp1c</i> level was comparable in <i>Tmed2</i>^<i>99J/+</i> and age-matched wildtype control mice at all stages. C). <i>Srebp2</i> level was increased in P5 <i>Tmed2</i>^<i>99J/+</i> mice compared to age-matched wildtype controls. D). Levels of activated SREBP1C was comparable in <i>Tmed2</i>^<i>99J/+</i> and age-matched wildtype control at 1–2 and 3–6 months. E). Levels of activated SREBP2 was comparable in <i>Tmed2</i>^<i>99J/+</i> and age-matched wildtype control at 1–2 and 3–6 months. F). Representative images of Western blot showing expression of full SREBP1C, cleaved SREBP1C (active form), SREBP2, and total protein loading control. 3 animals of each genotype were analyzed per age group. WT = wildtype. *P<0.05 by t-test.</p