Little is known about how patterns of DNA methylation change during mammalian spermatogenesis. 5hmC has been recognized as a stable intermediate of DNA demethylation with potential regulatory functions in the mammalian genome. However, its global pattern in germ cells has yet to be addressed. Here, we first conducted absolute quantification of 5hmC in eight consecutive types of mouse spermatogenic cells using liquid chromatographytandem mass spectrometry, and then mapped its distributions in various genomic regions using our chemical labeling and enrichment method coupled with deep sequencing. We found that 5hmC mapped differentially to and changed dynamically in genomic regions related to expression regulation of protein-coding genes, piRNA precursor genes and repetitive elements. Moreover, 5hmC content correlated with the levels of various transcripts quantified by RNA-seq. These results suggest that the highly ordered alterations of 5hmC in the mouse genome are potentially crucial for the differentiation of spermatogenic cells.

Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.

PIWI-interacting RNAs (piRNAs) are a class of small RNAs abundantly expressed in animal gonads. piRNAs that map to retrotransposons are generated by a ''ping-pong'' amplification loop to suppress the activity of retrotransposons. However, the biogenesis and function of other categories of piRNAs have yet to be investigated. In this study, we first profiled the expression of small RNAs in type A spermatogonia, pachytene spermatocytes, and round spermatids by deep sequencing. We then focused on the computational analysis of the potential piRNAs generated in the present study as well as other published sets. piRNAs mapping to retrotransposons, mRNAs, and intergenic regions had different length distributions and were differentially regulated in spermatogenesis. piRNA-generating mRNAs (PRMRs), whose expression positively correlated with their piRNA products, constituted one-third of the protein-coding genes and were evolutionarily conserved and enriched with splicing isoforms and antisense transcripts. PRMRs with piRNAs preferentially mapped to CDSs and 39 UTRs partitioned into three clusters differentially expressed during spermatogenesis and enriched with unique sets of functional annotation terms related to housekeeping activities as well as spermatogenesis-specific processes. Intergenic piRNAs were divided into 2992 clusters probably representing novel transcriptional units that have not been reported. The transcripts of a large number of genes involved in spermatogenesis are the precursors of piRNAs, and these genes are intricately regulated by alternative splicing and antisense transcripts. piRNAs, whose regulatory role in gene expression awaits to be identified, are clearly products of a novel regulatory process that needs to be defined.

In adaptating to perpetual darkness, cave species gradually lose eyes and body pigmentation and evolve alternatives for exploring their environments. Although troglodyte features evolved independently many times in cavefish, we do not yet know whether independent evolution of these characters involves common genetic mechanisms. Surface-dwelling and many cave-dwelling species make the freshwater teleost genus Sinocyclocheilus an excellent model for studying the evolution of adaptations to life in constant darkness. We compared the mature retinal histology of surface and cave species in Sinocyclocheilus and found that adult cavefish showed a reduction in the number and length of photoreceptor cells. To identify genes and genetic pathways that evolved in constant darkness, we used RNA-seq to compare eyes of surface and cave species. De novo transcriptome assemblies were developed for both species, and contigs were annotated with gene ontology. Results from cave-dwelling Sinocyclocheilus revealed reduced transcription of phototransduction and other genes important for retinal function. In contrast to the blind Mexican tetra cavefish Astyanax mexicanus, our results on morphologies and gene expression suggest that evolved retinal reduction in cave-dwelling Sinocyclocheilus occurs in a lens-independent fashion by the reduced proliferation and downregulation of transcriptional factors shown to have direct roles in retinal development and maintenance, including cone-rod homeobox (crx) and Wnt pathway members. These results show that the independent evolution of retinal degeneration in cavefish can occur by different developmental genetic mechanisms.

AbstractmiRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.

5-Hydroxymethylcytosine (5-hmC) may represent a new epigenetic modification of cytosine. While the dynamics of 5-hmC during neurodevelopment have recently been reported, little is known about its genomic distribution and function(s) in neurodegenerative diseases such as Huntington's disease (HD). We here observed a marked reduction of the 5-hmC signal in YAC128 (yeast artificial chromosome transgene with 128 CAG repeats) HD mouse brain tissues when compared with age-matched wild-type (WT) mice, suggesting a deficiency of 5-hmC reconstruction in HD brains during postnatal development. Genome-wide distribution analysis of 5-hmC further confirmed the diminishment of the 5-hmC signal in striatum and cortex in YAC128 HD mice. General genomic features of 5-hmC are highly conserved, not being affected by either disease or brain regions. Intriguingly, we have identified disease-specific (YAC128 versus WT) differentially hydroxymethylated regions (DhMRs), and found that acquisition of DhmRs in gene body is a positive epigenetic regulator for gene expression. Ingenuity pathway analysis (IPA) of genotype-specific DhMR-annotated genes revealed that alternation of a number of canonical pathways involving neuronal development/differentiation (Wnt/β-catenin/Sox pathway, axonal guidance signaling pathway) and neuronal function/survival (glutamate receptor/calcium/CREB, GABA receptor signaling, dopamine-DARPP32 feedback pathway, etc.) could be important for the onset of HD. Our results indicate that loss of the 5-hmC marker is a novel epigenetic feature in HD, and that this aberrant epigenetic regulation may impair the neurogenesis, neuronal function and survival in HD brain. Our study also opens a new avenue for HD treatment; re-establishing the native 5-hmC landscape may have the potential to slow/halt the progression of HD.

Mammalian spermatogenesis consists of many cell types and biological processes and serves as an excellent model for studying gene regulation at transcriptional and post-transcriptional levels. Many key proteins, miRNAs, and perhaps piRNAs have been shown to be involved in post-transcriptional regulation of spermatogenesis. However, a systematic method for assessing the relationship between protein and mRNA expression has not been available for studying mechanisms of post-transcriptional regulation. In the present study, we used the iTRAQbased quantitative proteomic approach to identify 2008 proteins in mouse type A spermatogonia, pachytene spermatocytes, round spermatids, and elongative spermatids with high confidence. Of these proteins, 1194 made up four dynamically changing clusters, which reflect the mitotic amplification, meiosis, and post-meiotic development of germ cells. We identified five major regulatory mechanisms termed "transcript only," "transcript degradation," "translation repression," "translation de-repression," and "protein degradation" based on changes in protein level relative to changes in mRNA level at the mitosis/meiosis transition and the meiosis/post-meiotic development transition. We found that post-transcriptional regulatory mechanisms are related to the generation of piRNAs and antisense transcripts. Our results provide a valuable inventory of proteins produced during mouse spermatogenesis and contribute to elucidating the mechanisms of the post-transcriptional regulation of gene expression in mammalian spermatogenesis. Molecular &

Among all tissues of the metazoa, the transcritpome of testis displays the highest diversity and specificity. However, its composition and dynamics during spermatogenesis have not been fully understood. Here, we have identified 20,639 message RNAs (mRNAs), 7,168 long non-coding RNAs (lncRNAs) and 15,101 circular RNAs (circRNAs) in mouse spermatogenic cells, and found many of them were specifically expressed in testes. lncRNAs are significantly more testis-specific than mRNAs. At all stages, mRNAs are generally more abundant than lncRNAs, and linear transcripts are more abundant than circRNAs. We showed that the productions of circRNAs and piRNAs were highly regulated instead of random processes. Based on the results of a small-scale functional screening experiment using cultured mouse spermatogonial stem cells, many evolutionarily conserved lncRNAs are likely to play roles in spermatogenesis. Typical classes of transcription factor binding sites are enriched in the promoters of testis-specific m/lncRNA genes. Target genes of CREM and RFX2, 2 key TFs for spermatogenesis, were further validated by using ChIP-chip assays and RNA-seq on RFX2-knockout spermatogenic cells. Our results contribute to the current understanding of the transcriptomic complexity of spermatogenic cells and provide a valuable resource from which many candidate genes may be selected for further functional studies.