Theresa V. Gibney

Summary: Apical constriction shapes tissues in fundamental events including gastrulation and neural tube formation. Diverse actin regulators play key roles in shaping cells, but the spatial distribution of most actin regulators in apical constriction is unknown. Here, we describe a systematic approach revealing the endogenous localization of 49 actin regulators in C. elegans gastrulation – nearly every actin-binding protein (ABP) expressed in the early embryo – and we use this atlas to test mechanistic hypotheses. We found that many actin regulators localized basolaterally, dependent on the Arp2/3 complex; that linear actin assembly factors enriched apically; and that apically constricting cells had an extra requirement for membrane-cortical linkers present in all cells. These findings answer some key questions about how ABPs function in apical constriction, and they establish a rich resource for investigating actin organization throughout early C. elegans development and a systematic in vivo protein localization reference for the actin cytoskeleton community.

Caenorhabditis elegans is a powerful model for dissecting biological processes in vivo. In particular, the ease of generating targeted knock-in alleles makes it possible to visualize and functionally modify endogenous proteins to gain fundamental insights into biological mechanisms. Methods for C. elegans genome engineering typically utilize selectable markers, visual screening for fluorescence, or PCR genotyping to identify successfully edited animals. A common genetic tool known as the self-excising cassette (SEC) combines drug and phenotypic selection, which makes it possible to screen large numbers of progeny rapidly and with minimal hands-on effort. However, N-terminal and internal knock-ins using the SEC cause loss of function until the selectable marker cassette is excised, which makes it impossible to isolate homozygous lines for essential genes prior to SEC excision. To simplify generating knock-ins for essential genes, we developed a nested, self-excising selection cassette (NSEC) that is located entirely within a synthetic intron and does not interfere with the expression of endogenously tagged NSEC-fusion proteins. This innovation makes it possible to isolate homozygous lines for N-terminally and internally tagged genes prior to selectable marker excision while preserving endogenous protein function. This method allows for a standardized workflow to generate N-terminal and internal tags in any background and without the need for genetic balancers. We designed versions of NSEC that include an optional auxin-inducible degron tag and mTurquoise2, GFP, mStayGold, mNeonGreen, or mScarlet-I fluorescent proteins for experimental flexibility. The NSEC expands our molecular toolbox and enhances the scalability, efficiency, and versatility of C. elegans genome engineering.

Extracellular signaling molecules mediate crucial aspects of cell–cell communication and play essential roles in development and homeostasis. Fibroblast growth factors (FGFs) are a family of secreted signaling proteins that can disperse long distances between cells and are often thought to form concentration gradients that encode spatial information. However, we know relatively little about the spatial distribution of FGFs in vivo, and endogenously tagged FGFs move between cells using different mechanisms in zebrafish and flies. We used FGF-dependent migration of C. elegans muscle progenitors called sex myoblasts (SMs) to elucidate FGF dispersal mechanisms and dissect how FGF guides migrating cells. Live imaging of cell dynamics and endogenously tagged FGF combined with membrane tethering and extracellular trapping approaches revealed that endogenous FGF is diffusible in vivo and extracellular dispersal is required for SM migration. Misexpression demonstrated that FGF is a bona fide chemoattractant that orients SMs during a critical window, while an unidentified, short-range signal acts in concert to position SMs precisely. Our finding that an invertebrate FGF is endogenously diffusible suggests that this may be the ancestral mode for FGF dispersal.

C. elegans is a powerful model for dissecting biological processes in vivo . In particular, the ease of generating targeted knock-in alleles makes it possible to visualize and functionally modify endogenous proteins to gain fundamental insights into biological mechanisms. Methods for C. elegans genome engineering typically utilize selectable markers, visual screening for fluorescence, or PCR genotyping to identify successfully edited animals. A common genetic tool known as the Self-Excising Cassette (SEC) combines drug and phenotypic selection, which makes it possible to screen large numbers of progeny rapidly and with minimal hands-on effort. However, N-terminal and internal knock-ins using the SEC cause loss of function until the selectable marker cassette is excised, which makes it impossible to isolate homozygous lines for essential genes prior to SEC excision. To simplify generating knock-ins for essential genes, we developed a Nested, Self-Excising selection Cassette (NSEC) that is located entirely within a synthetic intron and does not interfere with the expression of endogenous, N-terminally-tagged NSEC-fusion proteins. This innovation makes it possible to isolate homozygous lines for N-terminally tagged genes prior to selectable marker excision and allows for a standardized workflow to generate N-terminal and internal tags in any background and without the need for genetic balancers. We designed versions of NSEC that include an optional auxin-inducible degron tag and mTurquoise2, GFP, mStayGold, mNeonGreen, or mScarlet-I fluorescent proteins for experimental flexibility. The NSEC expands our molecular toolbox and enhances the scalability, efficiency, and versatility of C. elegans genome engineering.

Summary: Directed cell migration is essential for animal development, tissue maintenance, regeneration, and disease states. Cells often migrate towards, or away from, sources of secreted signaling proteins that impart spatial information. How migrating cells interpret extracellular signals to orient and navigate within living animals is a fundamental question in biology. Receptor Tyrosine Kinase (RTK) signaling plays critical roles in cell migration, and aberrant RTK pathway activity is a key driver of multiple types of cancers. Yet, how RTKs control cell migration in living animals remains unclear, in part due to essential, pleiotropic roles of key proteins in development. To elucidate how RTK signaling controls cell migration in vivo , we dissected spatial and temporal requirements for key signal transduction and cytoskeletal regulatory proteins using C. elegans muscle progenitor migration as a tractable model. Cell type-specific depletion of endogenously tagged proteins revealed that homologs of FGFR, GRB2, SOS, and Ras control cell migration independently of their canonical ERK, PI3K, Akt, mTOR, and PLCψ effectors. Instead, we found that FGF-dependent, polarized SOS-1 orients migrating cells towards an FGF source, and mislocalizing SOS activity within migrating cells severely disrupts migration independent of ERK. Cell type-specific, gain-of-function experiments demonstrated that activated Ras is largely permissive for anterior migration in this context, and an intragenic revertant identified in a screen for suppressors of activated Ras/let-60 revealed that signal transduction in migrating muscle progenitors can be genetically uncoupled from Ras-ERK-dependent developmental processes. We found that conserved regulators of branched actin assembly control SM protrusive dynamics but are not essential for accurate, FGF-directed migration. Our findings provide a novel mechanism for RTK-directed cell migration in vivo and highlight the importance of cell type-specific approaches to elucidate signal transduction mechanisms in physiologically relevant contexts. Our work also outlines a comprehensive framework for investigating RTK-dependent processes in a multicellular organism and introduces a versatile genetic toolkit for dissecting spatial and temporal signaling dynamics fundamental to development, homeostasis, and disease.

Extracellular signaling molecules mediate crucial aspects of cell-cell communication and play essential roles in development and homeostasis. Fibroblast Growth Factors (FGFs) are a conserved family of secreted signaling proteins that can disperse long distances between cells and are often thought to form concentration gradients that encode spatial information. However, we know relatively little about the spatial distribution of FGFs in vivo , and endogenously tagged FGFs use different mechanisms to move between cells and form gradients in zebrafish and flies. We used FGF-dependent migration of C. elegans muscle progenitors called sex myoblasts (SMs) as a tractable system to elucidate FGF dispersal mechanisms and dissect how FGF guides migrating cells. Live imaging of cell dynamics and endogenously tagged FGF combined with membrane tethering and extracellular trapping approaches revealed that endogenous FGF is diffusible in vivo and extracellular dispersal is required for SM migration. Misexpression demonstrated FGF is a bona fide chemoattractant that orients SMs during a critical window, while an unidentified, short-range signal acts in concert to precisely position SMs. Our finding that an invertebrate FGF is endogenously diffusible suggests that this may be the ancestral mode for FGF dispersal. Summary Statement: Fibroblast Growth Factors (FGFs) are signaling proteins with crucial developmental roles. We demonstrated that a C. elegans FGF homolog is diffusible, and diffusion is required in vivo for cell migration guidance.

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system (CNS) have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila CNS and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell-type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

Cell invasion through basement membrane (BM) barriers is important in development, immune function and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of Caenorhabditis elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including translationally controlled tumor protein (TCTP). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling and ribosome biogenesis analysis revealed that a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands before invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER-trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans . While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.