Genetic Mosaic Analysis Research Articles

Epithelial morphogenesis in the Drosophila egg chamber requires Parvin and ILK.

Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.

Fasciclin 2 engages EGFR in an auto-stimulatory loop to promote imaginal disc cell proliferation in Drosophila.

How cell to cell interactions control local tissue growth to attain a species-specific organ size is a central question in developmental biology. The Drosophila Neural Cell Adhesion Molecule, Fasciclin 2, is expressed during the development of neural and epithelial organs. Fasciclin 2 is a homophilic-interaction protein that shows moderate levels of expression in the proliferating epithelia and high levels in the differentiating non-proliferative cells of imaginal discs. Genetic interactions and mosaic analyses reveal a cell autonomous requirement of Fasciclin 2 to promote cell proliferation in imaginal discs. This function is mediated by the EGFR, and indirectly involves the JNK and Hippo signaling pathways. We further show that Fasciclin 2 physically interacts with EGFR and that, in turn, EGFR activity promotes the cell autonomous expression of Fasciclin 2 during imaginal disc growth. We propose that this auto-stimulatory loop between EGFR and Fasciclin 2 is at the core of a cell to cell interaction mechanism that controls the amount of intercalary growth in imaginal discs.

Mosaic gene expression analysis of semaphorin-plexin interactions in Caenorhabditis elegans using the IR-LEGO single-cell gene induction system.

Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism. However, the level of gene induction is not controllable due to the usage of a heat-shock promoter. Here, we applied IR-LEGO to examine the cell-cell interactions mediated by semaphoring-plexin signaling in Caenorhabditis elegans by inducing wild-type semaphorin/plexin in single cells within the population of mutant cells lacking the relevant proteins. We found that the cell contact-dependent termination of the extension of vulval precursor cells is elicited by the forward signaling mediated by the semaphorin receptor, PLX-1, but not by the reverse signaling via the transmembrane semaphorin, SMP-1. By utilizing Cre/loxP recombination coupled with the IR-LEGO system to induce SMP-1 at a physiological level, we found that SMP-1 interacts with PLX-1 only in trans upon contact between vulval precursor cells. In contrast, when overexpressed, SMP-1 exhibits the ability to cis-interact with PLX-1 on a single cell. These results indicate that mosaic analysis with IR-LEGO, especially when combined with an in vivo recombination system, efficiently complements conventional methods.

Ryanodine receptor 2 (RYR2) dysfunction activates the unfolded protein response and perturbs cardiomyocyte maturation.

Calcium-handling capacity is a major gauge of cardiomyocyte maturity. Ryanodine receptor 2 (RYR2) is the pre-dominant calcium channel that releases calcium from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) to activate cardiomyocyte contraction. Although RYR2 was previously implied as a key regulator of cardiomyocyte maturation, the mechanisms remain unclear. The aim of this study is to solve this problem. We performed Cas9/AAV9-mediated somatic mutagenesis to knockout RYR2 specifically in cardiomyocytes in mice. We conducted a genetic mosaic analysis to dissect the cell-autonomous function of RYR2 during cardiomyocyte maturation. We found that RYR2 depletion triggered ultrastructural and transcriptomic defects relevant to cardiomyocyte maturation. These phenotypes were associated with the drastic activation of ER stress pathways. The ER stress alleviator tauroursodeoxycholic acid partially rescued the defects in RYR2-depleted cardiomyocytes. Overexpression of ATF4, a key ER stress transcription factor, recapitulated defects in RYR2-depleted cells. Integrative analysis of RNA-Seq and bioChIP-Seq data revealed that protein biosynthesis-related genes are the major direct downstream targets of ATF4. RYR2-regulated ER homeostasis is essential for cardiomyocyte maturation. Severe ER stress perturbs cardiomyocyte maturation primarily through ATF4 activation. The major downstream effector genes of ATF4 are related to protein biosynthesis.

Cas9/AAV9-Mediated Somatic Mutagenesis Uncovered the Cell-Autonomous Role of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 in Murine Cardiomyocyte Maturation.

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) is a key player in cardiomyocyte calcium handling and also a classic target in the gene therapy for heart failure. SERCA2 expression dramatically increases during cardiomyocyte maturation in the postnatal phase of heart development, which is essential for the heart to acquire its full function in adults. However, whether and how SERCA2 regulates cardiomyocyte maturation remains unclear. Here, we performed Cas9/AAV9-mediated somatic mutagenesis (CASAAV) in mice and achieved cardiomyocyte-specific knockout of Atp2a2, the gene coding SERCA2. Through a cardiac genetic mosaic analysis, we demonstrated the cell-autonomous role of SERCA2 in building key ultrastructures of mature ventricular cardiomyocytes, including transverse-tubules and sarcomeres. SERCA2 also exerts a profound impact on oxidative respiration gene expression and sarcomere isoform switching from Myh7/Tnni1 to Myh6/Tnni3, which are transcriptional hallmarks of cardiomyocyte maturation. Together, this study uncovered a pivotal role of SERCA2 in heart development and provided new insights about SERCA2-based cardiac gene therapy.

A genome-wide library of MADM mice for single-cell genetic mosaic analysis.

Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions invivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.

Glutamate Signaling via the AMPAR Subunit GluR4 Regulates Oligodendrocyte Progenitor Cell Migration in the Developing Spinal Cord.

Oligodendrocyte progenitor cells (OPCs) are specified from discrete precursor populations during gliogenesis and migrate extensively from their origins, ultimately distributing throughout the brain and spinal cord during early development. Subsequently, a subset of OPCs differentiates into mature oligodendrocytes, which myelinate axons. This process is necessary for efficient neuronal signaling and organism survival. Previous studies have identified several factors that influence OPC development, including excitatory glutamatergic synapses that form between neurons and OPCs during myelination. However, little is known about how glutamate signaling affects OPC migration before myelination. In this study, we use in vivo, time-lapse imaging in zebrafish in conjunction with genetic and pharmacological perturbation to investigate OPC migration and myelination when the GluR4A ionotropic glutamate receptor subunit is disrupted. In our studies, we observed that gria4a mutant embryos and larvae displayed abnormal OPC migration and altered dorsoventral distribution in the spinal cord. Genetic mosaic analysis confirmed that these effects were cell-autonomous, and we identified that voltage-gated calcium channels were downstream of glutamate receptor signaling in OPCs and could rescue the migration and myelination defects we observed when glutamate signaling was perturbed. These results offer new insights into the complex system of neuron-OPC interactions and reveal a cell-autonomous role for glutamatergic signaling in OPCs during neural development.SIGNIFICANCE STATEMENT The migration of oligodendrocyte progenitor cells (OPCs) is an essential process during development that leads to uniform oligodendrocyte distribution and sufficient myelination for central nervous system function. Here, we demonstrate that the AMPA receptor (AMPAR) subunit GluR4A is an important driver of OPC migration and myelination in vivo and that activated voltage-gated calcium channels are downstream of glutamate receptor signaling in mediating this migration.

A Genome-Wide Library of MADM Mice for Single-Cell Genetic Mosaic Analysis

Mosaic Analysis with Double Markers (MADM) offers a unique approach to visualize and concomitantly manipulate genetically-defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage; single-cell morphology and physiology; genomic imprinting phenotypes; and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM could only be applied to 96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond proof-of-principle, we applied our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We found striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.

The Conserved IgSF9 Protein Borderless Regulates Axonal Transport of Presynaptic Components and Color Vision in Drosophila.

Normal brain function requires proper targeting of synaptic-vesicle (SV) and active-zone components for presynaptic assembly and function. Whether and how synaptogenic signals (e.g., adhesion) at axo-dendritic contact sites promote axonal transport of presynaptic components for synapse formation, however, remain unclear. In this study, we show that Borderless (Bdl), a member of the conserved IgSF9-family trans-synaptic cell adhesion molecules, plays a novel and specific role in regulating axonal transport of SV components. Loss of bdl disrupts axonal transport of SV components in photoreceptor R8 axons, but does not affect the transport of mitochondria. Genetic mosaic analysis, transgene rescue and cell-type-specific knockdown indicate that Bdl is required both presynaptically and postsynaptically for delivering SV components in R8 axons. Consistent with a role for Bdl in R8 axons, loss of bdl causes a failure of R8-dependent phototaxis response to green light. bdl interacts genetically with imac encoding for a member of the UNC-104/Imac/KIF1A-family motor proteins, and is required for proper localization of Imac in R8 presynaptic terminals. Our results support a model in which Bdl mediates specific axo-dendritic interactions in a homophilic manner, which upregulates the Imac motor in promoting axonal transport of SV components for R8 presynaptic assembly and function.SIGNIFICANCE STATEMENT Whether and how synaptogenic adhesion at axo-dendritic contact sites regulates axonal transport of presynaptic components remain unknown. Here we show for the first time that a trans-synaptic adhesion molecule mediates specific interactions at axo-dendritic contact sites, which is required for upregulating the UNC-104/Imac/KIF1A motor in promoting axonal transport of synaptic-vesicle components for presynaptic assembly and function.

Par3 is essential for the establishment of planar cell polarity of inner ear hair cells

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.

Dietary Restriction Improves Intestinal Cellular Fitness to Enhance Gut Barrier Function and Lifespan in D. Melanogaster

Loss of gut integrity is linked to various human diseases including inflammatory bowel disease. However, the mechanisms which lead to loss of barrier function remain poorly understood. Using D. melanogaster, we demonstrate that dietary restriction (DR) slows the age-related decline in intestinal integrity by enhancing enterocyte cellular fitness through upregulation of dMyc in the intestinal epithelium. Reduction of dMyc in enterocytes induces cell death through JNK signaling leading to increased gut permeability and reduced lifespan upon DR. Genetic mosaic and epistasis analyses suggest that cell competition, whereby neighboring cells eliminate unfit cells by apoptosis, mediates cell death in enterocytes with reduced levels of dMyc. Reducing enterocyte apoptosis partially rescued the increased gut permeability and shortened lifespan upon loss of dMyc. We propose that dMyc acts as a barometer of enterocyte cell fitness impacting intestinal homeostasis in response to changes in diet and age.

The mito::mKate2 mouse: A far-red fluorescent reporter mouse line for tracking mitochondrial dynamics in vivo.

Mitochondria are incredibly dynamic organelles that undergo continuous fission and fusion events to control morphology, which profoundly impacts cell physiology including cell cycle progression. This is highlighted by the fact that most major human neurodegenerative diseases are due to specific disruptions in mitochondrial fission or fusion machinery and null alleles of these genes result in embryonic lethality. To gain a better understanding of the pathophysiology of such disorders, tools for the in vivo assessment of mitochondrial dynamics are required. It would be particularly advantageous to simultaneously image mitochondrial fission-fusion coincident with cell cycle progression. To that end, we have generated a new transgenic reporter mouse, called mito::mKate2 that ubiquitously expresses a mitochondria localized far-red mKate2 fluorescent protein. Here we show that mito::mKate2 mice are viable and fertile and that mKate2 fluorescence can be spectrally separated from the previously developed Fucci cell cycle reporters. By crossing mito::mKate2 mice to the ROSA26R-mTmG dual fluorescent Cre reporter line, we also demonstrate the potential utility of mito::mKate2 for genetic mosaic analysis of mitochondrial phenotypes.

In vivo zebrafish morphogenesis shows Cyp26b1 promotes tendon condensation and musculoskeletal patterning in the embryonic jaw.

Integrated development of diverse tissues gives rise to a functional, mobile vertebrate musculoskeletal system. However, the genetics and cellular interactions that drive the integration of muscle, tendon, and skeleton are poorly understood. In the vertebrate head, neural crest cells, from which cranial tendons derive, pattern developing muscles just as tendons have been shown to in limb and trunk tissue, yet the mechanisms of this patterning are unknown. From a forward genetic screen, we determined that cyp26b1 is critical for musculoskeletal integration in the ventral pharyngeal arches, particularly in the mandibulohyoid junction where first and second arch muscles interconnect. Using time-lapse confocal analyses, we detail musculoskeletal integration in wild-type and cyp26b1 mutant zebrafish. In wild-type fish, tenoblasts are present in apposition to elongating muscles and condense in discrete muscle attachment sites. In the absence of cyp26b1, tenoblasts are generated in normal numbers but fail to condense into nascent tendons within the ventral arches and, subsequently, muscles project into ectopic locales. These ectopic muscle fibers eventually associate with ectopic tendon marker expression. Genetic mosaic analysis demonstrates that neural crest cells require Cyp26b1 function for proper musculoskeletal development. Using an inhibitor, we find that Cyp26 function is required in a short time window that overlaps the dynamic window of tenoblast condensation. However, cyp26b1 expression is largely restricted to regions between tenoblast condensations during this time. Our results suggest that degradation of RA by this previously undescribed population of neural crest cells is critical to promote condensation of adjacent scxa-expressing tenoblasts and that these condensations are subsequently required for proper musculoskeletal integration.

Evidence for a novel function of Awd in maintenance of genomic stability

The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. Here, we analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. We show that awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, our data demonstrate a novel function for awd in maintenance of genomic stability. Our results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd - induced genomic instability on tumour development and metastasis formation.

Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis

SummaryImproved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.

Heparan sulfate proteoglycans regulate autophagy in Drosophila

ABSTRACT Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.

Reduced Erg Dosage Impairs Survival of Hematopoietic Stem and Progenitor Cells.

ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Ergkd ) in which Erg expression can be conditionally restored by Cre recombinase. Ergkd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Ergkd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin- Sca-1+ c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Ergkd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785.

IDC2 and IDC3, two genes involved in cell non-autonomous signaling of fruiting body development in the model fungus Podospora anserina

Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus Podospora anserina. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures.

Radial glia regulate vascular patterning around the developing spinal cord.

Vascular networks surrounding individual organs are important for their development, maintenance, and function; however, how these networks are assembled remains poorly understood. Here we show that CNS progenitors, referred to as radial glia, modulate vascular patterning around the spinal cord by acting as negative regulators. We found that radial glia ablation in zebrafish embryos leads to excessive sprouting of the trunk vessels around the spinal cord, and exclusively those of venous identity. Mechanistically, we determined that radial glia control this process via the Vegf decoy receptor sFlt1: sflt1 mutants exhibit the venous over-sprouting observed in radial glia-ablated larvae, and sFlt1 overexpression rescues it. Genetic mosaic analyses show that sFlt1 function in trunk endothelial cells can limit their over-sprouting. Together, our findings identify CNS-resident progenitors as critical angiogenic regulators that determine the precise patterning of the vasculature around the spinal cord, providing novel insights into vascular network formation around developing organs.

A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis.

Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.