Differential isoform expression and alternative splicing in sex determination in mice

Planells, Benjamín; Gómez-Redondo, Isabel; Pericuesta, Eva; Lonergan, Patrick; Gutiérrez‐Adán, Alfonso

doi:10.1186/s12864-019-5572-x

Cited by 26 publications

(18 citation statements)

References 70 publications

(86 reference statements)

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“…Our study confirms and complements previous findings accumulated in humans and other species (Nef et al, 2005;Beverdam and Koopman, 2006;Jameson et al, 2012b;Zhao et al, 2018;Planells et al, 2019). For instance our dataset validates the onset of SRY transcription prior to 6 PCW in the testis as early as 6 PCW (previously reported at only 6.8 PCW in (Mamsen et al, 2017)), which may reflect the higher sensitivity of RNA-seq as compared to microarrays (Mantione et al, 2014).…”

Section: Distinct Cellular Expression Patterns Of Newly Identified Genes Involved In Human Sex Determinationsupporting

confidence: 93%

Dynamics of the transcriptional landscape during human fetal testis and ovary development

et al. 2020

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STUDY QUESTION Which transcriptional program triggers sex differentiation in bipotential gonads and downstream cellular events governing fetal testis and ovary development in humans? SUMMARY ANSWER The characterization of a dynamically regulated protein-coding and non-coding transcriptional landscape in developing human gonads of both sexes highlights a large number of potential key regulators that show an early sexually dimorphic expression pattern. WHAT IS KNOWN ALREADY Gonadal sex differentiation is orchestrated by a sexually dimorphic gene expression program in XX and XY developing fetal gonads. A comprehensive characterization of its non-coding counterpart offers promising perspectives for deciphering the molecular events underpinning gonad development and for a complete understanding of the etiology of disorders of sex development in humans. STUDY DESIGN, SIZE, DURATION To further investigate the protein-coding and non-coding transcriptional landscape during gonad differentiation, we used RNA-sequencing (RNA-seq) and characterized the RNA content of human fetal testis (N = 24) and ovaries (N = 24) from 6 to 17 postconceptional week (PCW), a key period in sex determination and gonad development. PARTICIPANTS/MATERIALS, SETTING, METHODS First trimester fetuses (6–12 PCW) and second trimester fetuses (13–14 and 17 PCW) were obtained from legally induced normally progressing terminations of pregnancy. Total RNA was extracted from whole human fetal gonads and sequenced as paired-end 2 × 50 base reads. Resulting sequences were mapped to the human genome, allowing for the assembly and quantification of corresponding transcripts. MAIN RESULTS AND THE ROLE OF CHANCE This RNA-seq analysis of human fetal testes and ovaries at seven key developmental stages led to the reconstruction of 22 080 transcripts differentially expressed during testicular and/or ovarian development. In addition to 8935 transcripts displaying sex-independent differential expression during gonad development, the comparison of testes and ovaries enabled the discrimination of 13 145 transcripts that show a sexually dimorphic expression profile. The latter include 1479 transcripts differentially expressed as early as 6 PCW, including 39 transcription factors, 40 long non-coding RNAs and 20 novel genes. Despite the use of stringent filtration criteria (expression cut-off of at least 1 fragment per kilobase of exon model per million reads mapped, fold change of at least 2 and false discovery rate adjusted P values of less than <1%), the possibility of assembly artifacts and of false-positive differentially expressed transcripts cannot be fully ruled out. LARGE-SCALE DATA Raw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI GEO under accession number GSE116278. LIMITATIONS, REASONS FOR CAUTION The intrinsic nature of this bulk analysis, i.e. the sequencing of transcripts from whole gonads, does not allow direct identification of the cellular origin(s) of the transcripts characterized. Potential cellular dilution effects (e.g. as a result of distinct proliferation rates in XX and XY gonads) may account for a few of the expression profiles identified as being sexually dimorphic. Finally, transcriptome alterations that would result from exposure to pre-abortive drugs cannot be completely excluded. Although we demonstrated the high quality of the sorted cell populations used for experimental validations using quantitative RT-PCR, it cannot be totally excluded that some germline expression may correspond to cell contamination by, for example, macrophages. WIDER IMPLICATIONS OF THE FINDINGS For the first time, this study has led to the identification of 1000 protein-coding and non-coding candidate genes showing an early, sexually dimorphic, expression pattern that have not previously been associated with sex differentiation. Collectively, these results increase our understanding of gonad development in humans, and contribute significantly to the identification of new candidate genes involved in fetal gonad differentiation. The results also provide a unique resource that may improve our understanding of the fetal origin of testicular and ovarian dysgenesis syndromes, including cryptorchidism and testicular cancers. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the French National Institute of Health and Medical Research (Inserm), the University of Rennes 1, the French School of Public Health (EHESP), the Swiss National Science Foundation [SNF n° CRS115_171007 to B.J.], the French National Research Agency [ANR n° 16-CE14-0017-02 and n° 18-CE14-0038-02 to F.C.], the Medical Research Council [MR/L010011/1 to P.A.F.] and the European Community’s Seventh Framework Programme (FP7/2007-2013) [under grant agreement no 212885 to P.A.F.] and from the European Union’s Horizon 2020 Research and Innovation Programme [under grant agreement no 825100 to P.A.F. and S.M.G.]. There are no competing interests related to this study.

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Section: Distinct Cellular Expression Patterns Of Newly Identified Genes Involved In Human Sex Determinationsupporting

confidence: 93%

Dynamics of the transcriptional landscape during human fetal testis and ovary development

et al. 2020

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“…We also identified 27 and 16 sex-specific genes in male and female PMRs that are also highly methylated in pig blastocysts (Additional files 5 and 6). Moreover, we found 156 and 143 genes containing PMRs in male and female germ cells, respectively, that are differentially expressed in male and female embryos during sex determination or express different isoforms in a sex-specific way in mice (Additional file 8) [44]. We also found that 45 of the genes containing PMRs identified in male germ cells and 48 in female germ cells are differentially expressed during sex determination in the cow (Additional file 8) [45].…”

Section: Demethylation-resistant Genomic Elements Are Candidate Players In Sex Determination and Early Differentiationmentioning

confidence: 75%

Genome-wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline

Gómez-Redondo¹,

Planells²,

Cánovas

et al. 2021

Clin Epigenet

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Background Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming. Results Sows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic-activated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage. Conclusions Our findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.

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“…Different isoforms of the same gene can regulate specific developmental and physiological processes (Graveley 2001; Baralle and Giudice 2017). Although alternative splicing has been shown to be important for sex determination in Drosophila (Burtis and Baker 1989) and for the development of sex-specific tissues in other species (Telonis-Scott et al 2009; Gibilisco et al 2016; Planells et al 2019), the overall importance of sex-specific alternative splicing remains largely underexplored (McIntyre et al 2006). This is because detecting full-length isoforms is difficult when using short read RNA-seq.…”

Section: Resultsmentioning

confidence: 99%

Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish

Shanfelter¹,

Pau

White

2020

Preprint

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Alternate isoforms contribute immensely to phenotypic diversity across eukaryotes. While short read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we utilize PacBio long read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five tissues in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly with gene annotations that are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq predicted transcription start sites were more accurate, verified through ATAC-seq. We were also able to detect many alternative splicing events between sexes and across tissues. We found a substantial number of genes in both somatic and gonad tissue that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.

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Differential isoform expression and alternative splicing in sex determination in mice

Cited by 26 publications

References 70 publications

Dynamics of the transcriptional landscape during human fetal testis and ovary development

Dynamics of the transcriptional landscape during human fetal testis and ovary development

Genome-wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline

Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish

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