Deep sequencing analysis of the developing mouse brain reveals a novel microRNA

Extent: 15p.Background: MicroRNAs (miRNAs) are small non-coding RNAs that can exert multilevel inhibition/repression at a post-transcriptional or protein synthesis level during disease or development. Characterisation of miRNAs in adult mammalian brains by deep sequencing has been reported previously. However, to date, no small RNA profiling of the developing brain has been undertaken using this method. We have performed deep sequencing and small RNA analysis of a developing (E15.5) mouse brain. Results: We identified the expression of 294 known miRNAs in the E15.5 developing mouse brain, which were mostly represented by let-7 family and other brain-specific miRNAs such as miR-9 and miR-124. We also discovered 4 putative 22-23 nt miRNAs: mm_br_e15_1181, mm_br_e15_279920, mm_br_e15_96719 and mm_br_e15_294354 each with a 70-76 nt predicted pre-miRNA. We validated the 4 putative miRNAs and further characterised one of them, mm_br_e15_1181, throughout embryogenesis. Mm_br_e15_1181 biogenesis was Dicer1-dependent and was expressed in E3.5 blastocysts and E7 whole embryos. Embryo-wide expression patterns were observed at E9.5 and E11.5 followed by a near complete loss of expression by E13.5, with expression restricted to a specialised layer of cells within the developing and early postnatal brain. Mm_br_e15_1181 was upregulated during neurodifferentiation of P19 teratocarcinoma cells. This novel miRNA has been identified as miR-3099. Conclusions: We have generated and analysed the first deep sequencing dataset of small RNA sequences of the developing mouse brain. The analysis revealed a novel miRNA, miR-3099, with potential regulatory effects on early embryogenesis, and involvement in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development.King-Hwa Ling, Peter J Brautigan, Christopher N Hahn, Tasman Daish, John R Rayner, Pike-See Cheah, Joy M Raison, Sandra Piltz Jeffrey R Mann, Deidre M Mattiske, Paul Q Thomas, David L Adelson and Hamish S Scot

Gata4 Is Required for Formation of the Genital Ridge in Mice

In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonadHoward Hughes Medical Institut

Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis

Prevalence of voice complaints, risk factors and impact of voice problems in female student teachers.

Item does not contain fulltextA cross-sectional questionnaire survey was done among 457 female student teachers and 144 females in the general population. The conclusions are based on the opinions of student teachers and the general population. The results of this study show that 39.6% of the student teachers and 32.6% of the general population reported voice complaints at the moment and/or over the past year (p=0.198). The association between various risk factors (vocal loading factors, physical factors, environmental factors and psycho-emotional factors) and voice complaints were examined. Vocal load was reported in both the student teachers and the general population (p=0.322). Among the subjects with voice complaints, the student teachers were significantly more of the opinion than the general population that environmental irritants in the classroom (p=0.001) and the composition of the group they communicate with (p=0.033) have a negative influence on their voice. In the groups with voice complaints, the student teachers reported significantly less than the general population that stress (p=0.004) and the deterioration of their general physical condition (p=0.003) have a negative influence on their voice. Remarkably, over a third of the student teachers and one fifth of the general population with voice complaints were of the opinion that decrease of hearing has a negative influence on their voices (p=0.113). There was no significant difference in Voice Handicap Index (VHI) scores (p=0.284) and impact of voice complaints among student teachers and the general population. Over 15% of the student teachers and the general population with voice complaints reported being or having been disabled due to the voice problem, probably reflecting the severity of the voice problem (p=0.838). The groups reporting voice complaints and disability in relation to their voice complaints have significantly higher VHI scores than those without voice complaints and disability, which indicates a higher psychosocial impact of voice complaints. Only around a third of the student teachers and the general population with voice complaints sought paramedical care (p=0.656)/treatment (p=0.361) for their voice complaint. Only a minority of student teachers (18.6%) and the general population (29.5%) with voice complaints were of the opinion that the number of people they communicate with has a negative influence on their voice (p=0.120). Only around a third of the student teachers and less than a tenth of the general population with voice complaints were of the view that they would develop a voice complaint due to their profession (p=0.003). Less than half of the student teachers and less than one fifth of the general population with voice complaints were aware of the potential risks of their profession on their voice (p=0.002). Voice complaints appear to have a multifactorial genesis. The student teachers are not sufficiently aware of the impact of the various risk factors on their voice. Furthermore, they are not aware of the potential risk that future teaching may have on their voice. This apparent lack of awareness in student teachers may be considered a risk factor for voice complaints

MicroRNAs and p63 in epithelial stemness

MicroRNAs (miRs) are a class of small noncoding RNAs that suppress the expression of protein-coding genes by repressing protein translation. Although the roles that miRs and the miR processing machinery have in regulating epithelial stem cell biology are not fully understood, their fundamental contributions to these processes have been demonstrated over the last few years. The p53-family member p63 is an essential transcription factor for epidermal morphogenesis and homeostasis. p63 functions as a determinant for keratinocyte cell fate and helps to regulate the balance between stemness, differentiation and senescence. An important factor that regulates p63 function is the reciprocal interaction between p63 and miRs. Some miRs control p63 expression, and p63 regulates the miR expression profile in the epidermis. p63 controls miR expression at different levels. It directly regulates the transcription of several miRs and indirectly regulates their processing by regulating the expression of the miR processing components Dicer and DGCR8. In this review, we will discuss the recent findings on the miR–p63 interaction in epidermal biology, particularly focusing on the ΔNp63-dependent regulation of DGCR8 recently described in the ΔNp63−/− mouse. We provide a unified view of the current knowledge and discuss the apparent discrepancies and perspective therapeutic opportunities