Cellular and molecular organization of the Drosophila foregut.

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.

Margarete Heck (1959-2023): Cell biologist, geneticist, and incandescent social spark.

Margarete M.S. Heck, professor of cell biology and genetics, University of Edinburgh, died peacefully at home amid her loving family under a blue moon on August 30, 2023, after a long journey with ovarian cancer.

The Ancient Origin and Function of Germline Cysts.

Gamete production in most animal species is initiated within an evolutionarily ancient multicellular germline structure, the germline cyst, whose interconnected premeiotic cells synchronously develop from a single progenitor arising just downstream from a stem cell. Cysts in mice, Drosophila, and many other animals protect developing sperm, while in females, cysts generate nurse cells that guard sister oocytes from transposons (TEs) and help them grow and build a Balbiani body. However, the origin and extreme evolutionary conservation of germline cysts remains a mystery.

Chromatin and gene expression changes during female germline stem cell development illuminate the biology of highly potent stem cells.

Highly potent animal stem cells either self renew or launch complex differentiation programs, using mechanisms that are only partly understood. female germline stem cells (GSCs) perpetuate without change over evolutionary time and generate cystoblast daughters that develop into nurse cells and oocytes. Cystoblasts initiate differentiation by generating a transient syncytial state, the germline cyst, and by increasing pericentromeric H3K9me3 modification, actions likely to suppress transposable element activity.

A symbiotic physical niche in Drosophila melanogaster regulates stable association of a multi-species gut microbiota.

The gut is continuously invaded by diverse bacteria from the diet and the environment, yet microbiome composition is relatively stable over time for host species ranging from mammals to insects, suggesting host-specific factors may selectively maintain key species of bacteria. To investigate host specificity, we used gnotobiotic Drosophila, microbial pulse-chase protocols, and microscopy to investigate the stability of different strains of bacteria in the fly gut. We show that a host-constructed physical niche in the foregut selectively binds bacteria with strain-level specificity, stabilizing their colonization.

Conservation of oocyte development in germline cysts from to mouse.

Recent studies show that pre-follicular mouse oogenesis takes place in germline cysts, highly conserved groups of oogonial cells connected by intercellular bridges that develop as nurse cells as well as an oocyte. Long studied in and insect gametogenesis, female germline cysts acquire cytoskeletal polarity and traffic centrosomes and organelles between nurse cells and the oocyte to form the Balbiani body, a conserved marker of polarity. Mouse oocyte development and nurse cell dumping are supported by dynamic, cell-specific programs of germline gene expression.

FMRP-dependent production of large dosage-sensitive proteins is highly conserved.

Mutations in FMR1 are the most common heritable cause of autism spectrum disorder. FMR1 encodes an RNA-binding protein, FMRP, which binds to long, autism-relevant transcripts and is essential for normal neuronal and ovarian development. In contrast to the prevailing model that FMRP acts to block translation elongation, we previously found that FMRP activates the translation initiation of large proteins in Drosophila oocytes.

Mouse oocytes develop in cysts with the help of nurse cells.

Mouse germline cysts, on average, develop into six oocytes supported by 24 nurse cells that transfer cytoplasm and organelles to generate a Balbiani body. We showed that between E14.5 and P5, cysts periodically activate some nurse cells to begin cytoplasmic transfer, which causes them to shrink and turnover within 2 days.

renal stem cells enhance fitness by delayed remodeling of adult Malpighian tubules.

renal stem cells (RSCs) contradict the common expectation that stem cells maintain tissue homeostasis. RSCs are abundant, quiescent, and confined to the peri-ureter region of the kidney-like Malpighian tubules (MTs). Although derived during pupation-like intestinal stem cells, RSCs initially remodel the larval MTs only near the intestinal junction.

High contiguity de novo genome assembly and DNA modification analyses for the fungus fly, Sciara coprophila, using single-molecule sequencing.

Background: The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery.

Altered germline cyst formation and oogenesis in Tex14 mutant mice.

During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization.

Differentiating female germ cells initiate Polycomb silencing by regulating PRC2-interacting proteins.

Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. We show that female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors.

Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary.

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.

An abundant quiescent stem cell population in Malpighian tubules protects principal cells from kidney stones.

Adult Malpighian tubules have low rates of cell turnover but are vulnerable to damage caused by stones, like their mammalian counterparts, kidneys. We show that renal stem cells (RSCs) in the ureter and lower tubules comprise a unique, unipotent regenerative compartment. RSCs respond only to loss of nearby principal cells (PCs), cells critical for maintaining ionic balance.

Prolonged ovarian storage of mature oocytes dramatically increases meiotic spindle instability.

Human oocytes frequently generate aneuploid embryos that subsequently miscarry. In contrast, oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, we developed a system to investigate how storage affects oocyte quality.

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.

We previously reported a CRISPR-mediated knock-in strategy into introns of genes, generating an transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms.

Dietary Lipids Modulate Notch Signaling and Influence Adult Intestinal Development and Metabolism in Drosophila.

Tissue homeostasis involves a complex balance of developmental signals and environmental cues that dictate stem cell function. We found that dietary lipids control enteroendocrine cell production from Drosophila posterior midgut stem cells. Dietary cholesterol influences new intestinal cell differentiation in an Hr96-dependent manner by altering the level and duration of Notch signaling.

Fragile X mental retardation 1 gene enhances the translation of large autism-related proteins.

Mutations in the fragile X mental retardation 1 gene () cause the most common inherited human autism spectrum disorder. FMR1 influences messenger RNA (mRNA) translation, but identifying functional targets has been difficult. We analyzed quiescent oocytes, which, like neural synapses, depend heavily on translating stored mRNA.

Efficient Expression of Genes in the Germline Using a UAS Promoter Free of Interference by Hsp70 piRNAs.

Controlling the expression of genes using a binary system involving the yeast GAL4 transcription factor has been a mainstay of developmental genetics for nearly 30 years. However, most existing GAL4 expression constructs only function effectively in somatic cells, but not in germ cells during oogenesis, for unknown reasons. A special upstream activation sequence (UAS) promoter, UASp was created that does express during oogenesis, but the need to use different constructs for somatic and female germline cells has remained a significant technical limitation.

A gene-specific library for .

We generated a library of ~1000 stocks in which we inserted a construct in the intron of genes allowing expression of under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes.

Polytene Chromosome Structure and Somatic Genome Instability.

Polytene chromosomes have for 80 years provided the highest resolution view of interphase genome structure in an animal cell nucleus. These chromosomes represent the normal genomic state of nearly all larval and many adult cells, and a better understanding of their striking banded structure has been sought for decades. A more recently appreciated characteristic of polytene cells is somatic genome instability caused by unfinished replication (UR).

The role of metabolic states in development and disease.

During development, cells adopt distinct metabolic strategies to support growth, produce energy, and meet the demands of a mature tissue. Some of these metabolic states maintain a constrained program of nutrient utilization, while others providing metabolic flexibility as a means to couple developmental progression with nutrient availability. Here we discuss our understanding of metabolic programs, and how they support specific aspects of animal development.

Identification of Genes Mediating Drosophila Follicle Cell Progenitor Differentiation by Screening for Modifiers of GAL4::UAS Variegation.

The Drosophila melanogaster ovarian follicle cell lineage provides a powerful system for investigating how epigenetic changes contribute to differentiation. Downstream from an epithelial stem cell, follicle progenitors undergo nine mitotic cell cycles before transitioning to the endocycle and initiating differentiation. During their proliferative phase, follicle progenitors experience Lsd1-dependent changes in epigenetic stability that can be monitored using GAL4::UAS variegation.

Single-Cell Lineage Analysis of Oogenesis in Mice.

Lineage analysis is widely used because it provides a very powerful tool for characterizing the developmental behavior of the cells in vivo. In this chapter, we describe a particularly informative variant of lineage analysis that we term "single-cell lineage analysis". As in traditional lineage analysis, the method employs a Tamoxifen (Tmx)-inducible CAGCreER mouse line, which is crossed to an R26R reporter line that can be activated by Cre-mediated DNA recombination.