Article-Journal

Maintaining genome integrity in germ cells is crucial for fertility and species survival. However, the DNA repair mechanisms that sustain genome integrity in primordial germ cells (PGCs), which cope with high levels of replication stress, remain largely unknown. While the TatD family of proteins, evolutionarily conserved nucleases, has been found to play roles in various DNA-related processes, their in vivo functions in vertebrates have yet to be fully elucidated. TATDN2 has recently been implicated in resolving R-loops and participating in the replication stress response in BRCA1-deficient cancer cells. Here we found that tatdn2 exhibits conserved expression in mitotic and early meiotic germ cells across teleosts and mammals. Using medaka fish as a model, we then showed that loss of tatdn2 leads to all-phenotypically male adults and infertility due to PGC depletion during mitotic proliferation. We further demonstrated that knockout of tatdn2 increases R-loop accumulation and DNA damage, subsequently triggering apoptosis in PGCs. These findings indicate that tatdn2 plays a critical role in DNA damage repair associated with R-loop resolution in mitotic PGCs. Our study provides novel insights into the physiological function of TATDN2 and the mechanisms of genome maintenance in PGCs.

Abstract: The ENCODE project provides a comprehensive map of genomic activity in humans and mice. However, its extensive toolbox can be too costly and technically challenging for smaller research communities. This presents a hurdle for understudied model organisms where resource constraints and complex omics data integration further compound the challenge. To bridge this gap, our study introduces a miniENCODE project, using the zebrafish as an exemplar. We collected, generated and integrated datasets from three experimental assays across developmental stages and adult tissues. This analysis is facilitated by our newly developed miniODP database, a computational tool designed for the integration and visualization of multi-omics data. Utilizing these methods, we have cataloged over 52,000 candidate enhancers, identified various stage-specific, tissue-specific, and constantly active enhancers, and experimentally validated a subset of heart-specific enhancers. We have delineated key transcription factors and their corresponding regulatory networks across developmental stages and adult tissues. This study not only delivers valuable regulatory insights for the zebrafish research community but also illustrates how an integrative approach can aid in understanding complex regulatory mechanisms even with limited resources. This strategy could empower scientific communities working with various understudied model organisms to expedite their genomic research without overstretching their resources.Competing Interest StatementThe authors have declared no competing interest.

Abstract: The mitotic quiescence of prospermatogonia is the event known to occur during genesis of the male germline and is tied to the development of the spermatogenic lineage. The regulatory mechanisms and the functional importance of this process have been demonstrated in mice; however, regulation of this process in human and domestic animal is still largely unknown. In this study, we employed single-cell RNA sequencing to identify transcriptional signatures of prospermatogonia and major somatic cell types in testes of goats at E85, E105, and E125. We identified both common and specific Gene Ontology categories, transcription factor regulatory networks, and cell-cell interactions in cell types from goat testis. We also analyzed the transcriptional dynamic changes in prospermatogonia, Sertoli cells, Leydig cells, and interstitial cells. Our datasets provide a useful resource for the study of domestic animal germline development.

Abstract: The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of Nuclear Factor I (NFI) family that was initially purified in host cells to promote adenoviral DNA replication but has since mainly been investigated in transcription regulation, is physically associated with the pre-replication complex (pre-RC) in mammalian cells. Genomic analyses reveal that NFIB facilitates the assembly of the pre-RC by increasing chromatin accessibility. Nucleosome binding and single-molecule magnetic tweezers shows that NFIB binds to and opens up nucleosomes. Transmission electron microscopy indicates that NFIB promotes nucleosome eviction on parental chromatin. NFIB deficiency leads to alterations of chromosome contacts/compartments in both G1 and S phase and affects the firing of a subset of origins at early-replication domains. Significantly, cancer-associated NFIB overexpression provokes gene duplication and genomic alterations recapitulating the genetic aberrance in clinical breast cancer and empowering cancer cells to dynamically evolve growth advantage and drug resistance. Together, these results point a role for NFIB in facilitating replication licensing by acting as a genome organizer, shedding new lights on the biological function of NFIB and on the replication origin selection in eukaryotes.

Abstract: Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart.

Abstract: The differences in size and function between primate and rodent brains, and the association of disturbed excitatory/inhibitory balance with many neurodevelopmental disorders highlight the importance to study primate ganglionic eminences (GEs) development. Here we used single-cell RNA and ATAC sequencing to characterize the emergence of cell diversity in monkey and human GEs where most striatal and cortical interneurons are generated. We identified regional and temporal diversity among progenitor cells which give rise to a variety of interneurons. These cells are specified within the primate GEs by well conserved gene regulatory networks, similar to those identified in mice. However, we detected, in human, several novel regulatory pathways or factors involved in the specification and migration of interneurons. Importantly, comparison of progenitors between our human and published mouse GE datasets led to the discovery and confirmation of outer radial glial cells in GEs in human cortex. Our findings reveal both evolutionarily conservative and nonconservative regulatory networks in primate GEs, which may contribute to their larger brain sizes and more complex neural networks compared with mouse.

Abstract: Successful pregnancy in placental mammals substantially depends on the establishment of maternal immune tolerance to the semi-allogenic fetus. Disorders in this process are tightly associated with adverse pregnancy outcomes including recurrent miscarriage (RM). However, an in-depth understanding of the systematic and decidual immune environment in RM remains largely lacking. In this study, we utilized single-cell RNA-sequencing (scRNA-seq) to comparably analyze the cellular and molecular signatures of decidual and peripheral leukocytes in normal and unexplained RM pregnancies at the early stage of gestation. Integrative analysis identifies 22 distinct cell clusters in total, and a dramatic difference in leukocyte subsets and molecular properties in RM cases is revealed. Specifically, the cytotoxic properties of CD8+ effector T cells, nature killer (NK), and mucosal-associated invariant T (MAIT) cells in peripheral blood indicates apparently enhanced pro-inflammatory status, and the population proportions and ligand-receptor interactions of the decidual leukocyte subsets demonstrate preferential immune activation in RM patients. The molecular features, spatial distribution, and the developmental trajectories of five decidual NK (dNK) subsets have been elaborately illustrated. In RM patients, a dNK subset that supports embryonic growth is diminished in proportion, while the ratio of another dNK subset with cytotoxic and immune-active signature is significantly increased. Notably, a unique pro-inflammatory CD56+CD16+ dNK subset substantially accumulates in RM decidua. These findings reveal a comprehensive cellular and molecular atlas of decidual and peripheral leukocytes in human early pregnancy and provide an in-depth insight into the immune pathogenesis for early pregnancy loss.

Abstract: Medaka (Oryzias latipes) has become an important vertebrate model widely used in genetics, developmental biology, environmental sciences, and many other fields. A high-quality genome sequence and a variety of genetic tools are available for this model organism. However, existing genome annotation is still rudimentary, as it was mainly based on computational prediction and short-read RNA-seq data. Here we report a dynamic transcriptome landscape of medaka embryogenesis profiled by long-read RNA-seq, short-read RNA-seq, and ATAC-seq. Integrating these datasets, we constructed a much-improved gene model set including about 17,000 novel isoforms, identified 1600 transcription factors, 1100 long non-coding RNAs, and 150,000 potential cis-regulatory elements as well. Time-series datasets provided another dimension of information. With the expression dynamics of genes and accessibility dynamics of cis-regulatory elements, we investigated isoform switching, regulatory logic between accessible elements and genes during embryogenesis. We built a user-friend medaka omics data portal to present these datasets. This resource provides the first comprehensive omics datasets of medaka embryogenesis. Ultimately, we term these three assays as the minimum ENCODE toolbox and propose the use of it as the initial and essential profiling genomic assays for model organisms that have limited data available. This work will be of great value for the research community using medaka as the model organism and many others as well.

Abstract: Many differential gene expression analyses are conducted with an inadequate number of biological replicates. We describe an easy and effective RNA-seq approach using molecular barcoding to enable profiling of a large number of replicates simultaneously. This approach significantly improves the performance of differential gene expression analysis. Using this approach in medaka (Oryzias latipes), we discover novel genes with sexually dimorphic expression and genes necessary for germ cell development. Our results also demonstrate why the common practice of using only three replicates in differential gene expression analysis should be abandoned.