Transient chromatin decompaction at the start of D. melanogaster male embryonic germline development.

Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.

Evidence for parallel evolution of a gene involved in the regulation of spermatogenesis.

PHD finger protein 7 (Phf7) is a male germline specific gene in Drosophila melanogaster that can trigger the male germline sexual fate and regulate spermatogenesis, and its human homologue can rescue fecundity defects in male flies lacking this gene. These findings prompted us to investigate conservation of reproductive strategies through studying the evolutionary origin of this gene. We find that Phf7 is present only in select species including mammals and some insects, whereas the closely related G2/M-phase specific E3 ubiquitin protein ligase (G2e3) is in the genome of most metazoans. Interestingly, phylogenetic analyses showed that vertebrate and insect Phf7 genes did not evolve from a common Phf7 ancestor but rather through independent duplication events from an ancestral G2e3 This is an example of parallel evolution in which a male germline factor evolved at least twice from a pre-existing template to develop new regulatory mechanisms of spermatogenesis.

Identification of low-molecular-weight vitellogenin 1 (Vg1)-like proteins as nucleotide excision repair (NER) factors in developing zebrafish (Danio rerio) using a transcription-based DNA repair assay.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions such as UV-induced pyrimidine dimers and cisplatin-induced strand crosslinking. Our earlier studies have identified low-molecular-weight proteins homologous to the 150-kDa vitellogenin 1 (Vg1) as UV-damaged DNA-binding factors expressed in developing zebrafish (Danio rerio). This present study explored if Vg1-like proteins also participated in NER in zebrafish. Immunoblot analysis of affinity-captured 12 h post-fertilization (hpf) zebrafish extract proteins showed a transient binding of a 30-kDa Vg1-like polypeptide to UV-damaged DNA. A transcription-based in vitro repair assay revealed a significant up-regulation of UVC or cisplatin-suppressed transcriptional activity of a marker cDNA driven by a SP6 RNA polymerase-regulated promotor after incubating the damaged plasmid with the extracts of 12 hpf embryos or 96 hpf larvae. The up-regulation of UV or cisplatin-suppressed transcription was abolished in the presence of a monoclonal anti-zebrafish Vg1 antibody. The differential sensitivity of UV-induced repair in 12 and 96 hpf zebrafish extracts to exogenous ATP suggested a development-dependent expression of Vg1-like NER factors. A T4 endonuclease V digestion assay showed no inhibition of the anti-Vg1 antibody on the excision of UV-induced cyclobutane pyrimidine dimers. Our results identified the participation of Vg1-like factors in NER in developing zebrafish, and these factors may function at post-incison steps of NER.

Oxidative stress intensity-related effects of cadmium (Cd) and paraquat (PQ) on UV-damaged-DNA binding and excision repair activities in zebrafish (Danio rerio) embryos.

Our earlier studies showed the inhibitory effects of cadmium (Cd) and paraquat (PQ) on the gene expression of DNA mismatch recognition proteins in zebrafish (Danio rerio) embryos. This study explored the effects of Cd and PQ on nucleotide excision repair (NER) capacity in zebrafish embryos. Exposure of embryos at 1 h post fertilization (hpf) to 3-5 µM Cd or 30-100 µM PQ for 9 h induced a 2-3-fold increase of oxidative stress, while a 6.5-fold increase of oxidative stress was induced by 200 µM PQ. Real-time RT-PCR detected a down-regulated xeroderma pigmentosum C (XPC) and an up-regulated UV-DDB2 gene expression in mildly-stressed embryos, whereas 8-oxoguanine DNA glycosylase (OGG1) gene expression increased with PQ exposure levels. NER of UV-damaged DNA was enhanced in weakly oxidant-stressed embryos as shown by a transcription-based DNA repair assay, yet repair activities of both UV and cisplatin-damaged DNA were inhibited in embryos exposed to 200 µM PQ. Band shift assay showed a suppression of cyclobutane pyrimidine dimer (CPD) binding activity in all stressed embryos. In contrast, (6-4) photoproduct (6-4PP) recognition activity was weakly stimulated except in embryos exposed to 200 µM PQ, revealing a link of NER capacity to 6-4PP binding. Our results showed that Cd and PQ imposed similar inducing effects on UV-DDB2 gene expression, NER of UV-damaged DNA and 6-4PP binding activity in zebrafish embryo under low levels of oxidative stress and NER capacity could be inhibited if the intensity of oxidative stress increased to a critical level.