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The animal foregut is the first tissue to encounter ingested food, bacteria, and viruses. We characterized the adult Drosophila foregut using transcriptomics to better understand how it triages consumed items for digestion or immune response and manages resources. Cell types were assigned and validated using GFP-tagged and Gal4 reporter lines. Foregut-associated neuroendocrine cells play a major integrative role by coordinating gut activity with nutrition, the microbiome, and circadian cycles; some express clock genes. Multiple epithelial cell types comprise the proventriculus, the central foregut organ that secretes the peritrophic matrix (PM) lining the gut. Analyzing cell types synthesizing individual PM layers revealed abundant mucin production close to enterocytes, similar to the mammalian intestinal mucosa. The esophagus and salivary gland express secreted proteins likely to line the esophageal surface, some of which may generate a foregut commensal niche housing specific gut microbiome species. Overall, our results imply that the foregut coordinates dietary sensing, hormonal regulation, and immunity in a manner that has been conserved during animal evolution. [ABSTRACT FROM AUTHOR]

Stem and embryonic cells facilitate programming toward multiple daughter cell fates, whereas differentiated cells resist reprogramming and oncogenic transformation. How alterations in the chromatin-based machinery of epigenetic inheritance contribute to these differences remains poorly known. We observed random, heritable changes in GAL4/UAS transgene programming during Drosophila ovarian follicle stem cell differentiation and used them to measure the stage-specific epigenetic stability of gene programming. The frequency of GAL4/UAS reprogramming declines more than 100-fold over the nine divisions comprising this stem cell lineage. Stabilization acts in cis, suggesting that it is chromatin-based, and correlates with increased S phase length. Our results suggest that stem/early progenitor cells cannot accurately transmit nongenetic information to their progeny; full epigenetic competence is acquired only gradually during early differentiation. Modulating epigenetic inheritance may be a criticai process controlling transitions between the pleuripotent and differentiated states. epigenetic inheritance | pleuripotency | cell lineage | reprogramming doi/ 10.1073/pnas.1003180107

The Balbiani body or mitochondrial cloud is a large distinctive organelle aggregate found in developing oocytes of many species, but its presence in the mouse has been controversial. Using confocal and electron microscopy, we report that a Balbiani body does arise in mouse neonatal germline cysts and oocytes of primordial follicles but disperses as follicles begin to grow. The mouse Balbiani body contains a core of Golgi elements surrounded by mitochondria and associated endoplasmic reticulum. Because of their stage specificity and perinuclear rather than spherical distribution, these clustered Balbiani body mitochondria may have been missed previously. The Balbiani body also contains Trailer hitch, a widely conserved member of a protein complex that associates with endoplasmic reticulum/Golgi-like vesicles and transports specific RNAs during Drosophila oogenesis. Our results provide evidence that mouse oocytes develop using molecular and developmental mechanisms widely conserved throughout the animal kingdom. germ plasm | gamete biology | oocyte development | ovary | oogenesis

Lethal alleles of the Drosophila k43 gene result in small or missing imaginal discs, greatly reduced mitotic index, and fragmented and abnormally condensed chromosomes. A female-sterile allele of k43 specifically reduces chorion gene amplification in ovarian follicle cells. k43 was cloned by chromosomal walking, and the identification of the k43 gene was confirmed by phenotypic rescue and sequence analysis of mutant alleles. The sequence analyses reveal that the k43 gene encodes the Drosophila homolog of the yeast origin recognition complex subunit 2 (Orc2p), a protein required for replication origin function and transcriptional silencing in yeast. These results suggest an evolutionarily conserved role for Orc2p in eukaryotic chromosomal DNA replication.

Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking, the insertions is sequenced, thereby placing an extensive series of genetic markeis on the physical genomic map and associating insertions with specific open reading frames and genes. Insertions from the collection now lie within or near most Drosophila genes, greatly reducing the time required to identify new mutations and analyze gene functions. Information revealed from these studies about P element site specificity is being used to target the remaining open reading frames.

A study of correction events within heterochromatic repeated DNA, associated with double-strand excisions initiated by transposons, involves acceleration of genetically marked elements present in a subtelomeric tandem repeat and confirms that the transposable P elements from tandemly repeated DNA initiate nonuniform gene conversion. Gene conversion is associated with varying tandem repeats where repeat decreases are more favored than increases due to a conversion bias. Inherent processes that monitor the double-strand excision repair lead to the conversion bias.

Insertional mutagenesis with transposable P elements were used to identify and evaluate genes located throughout the Drosophila melanogaster genome that was classified as euchromatin. Strains containing insertions at diverse sites were generated within centromeric and y-chromosome heterochromatin. Drosophila euchromatin was divided in polytene. Chromosomes in 5000 regions averaged 25 kilobase in length.

We sequenced more than 52,500 single cells from embryonic day 11.5 (E11.5) postembryonic day 5 (P5) gonads and performed lineage tracing to analyze primordial follicles and wave 1 medullar follicles during mouse fetal and perinatal oogenesis. Germ cells clustered into six meiotic substages, as well as dying/nurse cells. Wnt-expressing bipotential precursors already present at E11.5 are followed at each developmental stage by two groups of ovarian pregranulosa (PG) cells. One PG group, bipotential pregranulosa (BPG) cells, derives directly from bipotential precursors, expresses Foxl2 early, and associates with cysts throughout the ovary by E12.5. A second PG group, epithelial pregranulosa (EPG) cells, arises in the ovarian surface epithelium, ingresses cortically by E12.5 or earlier, expresses Lgr5, but delays robust Foxl2 expression until after birth. By E19.5, EPG cells predominate in the cortex and differentiate into granulosa cells of quiescent primordial follicles. In contrast, medullar BPG cells differentiate along a distinct pathway to become wave 1 granulosa cells. Reflecting their separate somatic cellular lineages, second wave follicles were ablated by diptheria toxin treatment of Lgr5-DTR-EGFP mice at E16.5 while first wave follicles developed normally and supported fertility. These studies provide insights into ovarian somatic cells and a resource to study the development, physiology, and evolutionary conservation of mammalian ovarian follicles. [ABSTRACT FROM AUTHOR]

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo. [ABSTRACT FROM AUTHOR]

The P transposable element recently invaded wild Drosophila melanogaster strains worldwide. A single introduced copy can multiply and spread throughout the fly genome in just a few generations. even though its cut-and-paste transposition mechanism does not inherently increase copy number. P element insertions preferentially target the promoters of a subset of genes, but why these sites are hotspots remains unknown. We show that P elements selectively target sites that in tissue-culture cells bind origin recognition complex proteins and function as replication origins. The association of origin recognition complex-binding sites with selected promoters and their absence near clustered differentiation genes may dictate P element site specificity. Inserting at unfired replication origins during S phase may allow P elements to be both repaired and reduplicated, thereby increasing element copy number. The advantage transposons gain by moving from replicated to unreplicated genomic regions may contribute to the association of heterochromatin with late-replicating genomic regions. [ABSTRACT FROM AUTHOR]

The article presents the author's opinion on the need for the U.S. National Institutes of Health (NIH) to support broadly focused basic research. The author notes the continued recognition of the critical role of basic research. The author suggests that all who are ivolved in the scientific community should support and help to accomplish the job.

The animal foregut is the first tissue to encounter ingested food, bacteria, and viruses. We characterized the adult Drosophila foregut using transcriptomics to better understand how it triages consumed items for digestion or immune response and manages resources. Cell types were assigned and validated using GFP-tagged and Gal4 reporter lines. Foregut-associated neuroendocrine cells play a major integrative role by coordinating gut activity with nutrition, the microbiome, and circadian cycles; some express clock genes. Multiple epithelial cell types comprise the proventriculus, the central foregut organ that secretes the peritrophic matrix (PM) lining the gut. Analyzing cell types synthesizing individual PM layers revealed abundant mucin production close to enterocytes, similar to the mammalian intestinal mucosa. The esophagus and salivary gland express secreted proteins likely to line the esophageal surface, some of which may generate a foregut commensal niche housing specific gut microbiome species. Overall, our results imply that the foregut coordinates dietary sensing, hormonal regulation, and immunity in a manner that has been conserved during animal evolution., Competing Interests: Competing interests statement:The authors declare no competing interest.

We sequenced more than 52,500 single cells from embryonic day 11.5 (E11.5) postembryonic day 5 (P5) gonads and performed lineage tracing to analyze primordial follicles and wave 1 medullar follicles during mouse fetal and perinatal oogenesis. Germ cells clustered into six meiotic substages, as well as dying/nurse cells. Wnt-expressing bipotential precursors already present at E11.5 are followed at each developmental stage by two groups of ovarian pregranulosa (PG) cells. One PG group, bipotential pregranulosa (BPG) cells, derives directly from bipotential precursors, expresses Foxl2 early, and associates with cysts throughout the ovary by E12.5. A second PG group, epithelial pregranulosa (EPG) cells, arises in the ovarian surface epithelium, ingresses cortically by E12.5 or earlier, expresses Lgr5, but delays robust Foxl2 expression until after birth. By E19.5, EPG cells predominate in the cortex and differentiate into granulosa cells of quiescent primordial follicles. In contrast, medullar BPG cells differentiate along a distinct pathway to become wave 1 granulosa cells. Reflecting their separate somatic cellular lineages, second wave follicles were ablated by diptheria toxin treatment of Lgr5-DTR-EGFP mice at E16.5 while first wave follicles developed normally and supported fertility. These studies provide insights into ovarian somatic cells and a resource to study the development, physiology, and evolutionary conservation of mammalian ovarian follicles., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)