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Gordon, K. L.

Publications and source records attributed to Gordon, K. L..

7 recordsLinked to original sources

FLYWCH transcription factors act in a LIN-42/Period autoregulatory loop during gonad migration in C. elegans

Development must be coordinated across body systems but must also accommodate cell-type-specific processes. We discovered that the gene regulatory circuit controlling developmental timing in the Caenorhabditis elegans larval skin exhibits both points of convergence and divergence with the regulatory program governing the migration of the leader cell in gonad development, the distal tip cell (DTC). As a point of convergence, the conserved regulator of developmental timing, LIN-42/Period, peaks synchronously across cell types both during the L3 stage, when the DTC makes a turn in its normal migratory path, and the L4 stage in which the DTC normally continues straight ahead. We report that lin-42, like its ortholog period, autorepresses its own transcription. lin-42 is required cell-autonomously for proper pathfinding of the DTC; DTC-specific lin-42 RNAi causes the DTC to turn in the mid-L4 instead of continuing straight ahead. We identified the FLYWCH transcription factor FLH-1 as able to directly bind the lin-42a promoter. Using live-cell imaging, we show that flh-1; flh-2 double mutant DTCs have an aberrant turn in the mid-L4. These mutants derepress the L4 peak of lin-42 expression in a stage- and DTC-specific manner, and this derepression is itself lin-42-dependent. During the aberrant mid-L4 turn in flh-1; flh-2 mutants, the focal adhesion factor TLN-1 is repolarized in the direction of turning. These results reveal that bodywide developmental rhythms can be fine-tuned to integrate with specific organogenic processes. SUMMARY STATEMENTTo identify new transcriptional regulators of distal tip cell migration, a screen for C. elegans transcription factors that bind the promoter of the Period homolog lin-42, a conserved regulator of developmental timing, uncovers a pair of factors that redundantly prevent misdirected migration of the distal tip cell-the cell that guides gonadal development. Through phenotypic analysis of genetic mutants, RNAi knockdown, and live imaging, we found that these transcription factors create a cell-specific autoregulatory loop that controls lin-42 transcription and promotes directional migration by polarizing the focal adhesion protein TLN-1.

developmental biology↗

Pre-dauer starvation decouples somatic from germline development with lifelong reproductive consequences in C. elegans

Early life stresses impact reproductive outcomes in many organisms. In response to crowding and starvation, C. elegans nematodes form dauer larvae, in which development arrests until conditions improve. We discovered dramatic differences in gonad size and germ cell number among dauers that form under different conditions. We used live cell imaging of fluorescent proteins in otherwise wild-type and mutant animals combined with food-removal, recovery, and brood-size assays to investigate the causes and consequences of this germline difference. Pre-dauer feeding, but not nutrient sensing via the DAF-2/insulin-like signaling receptor or DAF-7/TGF-{beta}, is required for plasticity in gonad size. Gonad differences in dauer have lifelong reproductive consequences; severely starved worms make small dauer gonads and have small broods. Pre-dauer starvation induces germline quiescence and near-instantaneous reduction of the Notch ligand LAG-2 on the germline stem cell niche. A rapid return to germline Notch dependence and an increase in presentation by the germline stem cell niche of LAG-2-independent of lag-2 transcriptional upregulation-are among the earliest events of dauer recovery. SUMMARY STATEMENTPre-dauer starvation arrests gonad growth, reduces presentation of the germline niche cue LAG-2, and diminishes future reproductive success. Dauer recovery rapidly restores LAG-2 ligand and germline Notch dependence.

developmental biology↗

MIG-21 is a novel regulator of Wnt and Netrin signaling in gonad migration identified from published scRNA-seq data and functionally validated in C. elegans

Using a recently published scRNA-seq dataset of adult C. elegans hermaphrodites, we identified a previously unknown regulator of the germ line stem cell niche (the distal tip cell, or DTC). The gene mig-21 has the highest "marker score"-yet no known role-in the DTC. Using classical genetics techniques, RNAi knockdown, and live cell imaging, we discovered that mig-21 integrates information from the Wnt and Netrin pathways to guide anteroposterior and dorsoventral DTC migration. Our study demonstrates the utility of scRNA-seq datasets in revealing testable hypotheses about genetic networks that were masked by redundancy in traditional screening methods.

developmental biology↗

Getting there in one piece: The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during organogenesis

The C. elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C. elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and in vivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation--the now-smaller DTC and the membranous patch of cellular material--localize important stem cell niche signaling (LAG-2/DSL ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long-term. Prior work found that Rac1-/-;Rac2-/-mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.

developmental biology↗

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

cell biology↗

The C. elegans gonadal sheath Sh1 cells associate selectively with a differentiating germ cell population in the proliferative zone

The C. elegans adult hermaphrodite germ line is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis and ovulation. Recently, we discovered that the distal Sh1 sheath cells associate with mitotic germ cells as they exit the niche (Gordon et al., 2020). Here we report that these sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other.

developmental biology↗

HIV antiretrovirals have differential effects on viability and function in human iPSC-derived neurons and neural precursor cells

Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that certain antiretrovirals (ARVs) may contribute to HAND. We examined the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG) on viability, structure, and function of glutamatergic neurons (a subtype of CNS neuron involved in cognition) derived from human induced pluripotent stem cells (hiPSC-neurons), and primary human neural precursor cells (hNPCs), which are responsible for neurogenesis. Using automated digital microscopy and image analysis (high content analysis, HCA), we found that DTG, EVG, and TDF decreased hiPSC-neuron viability, neurites, and synapses after seven days of treatment. Analysis of hiPSC-neuron calcium activity using Kinetic Image Cytometry (KIC) demonstrated that DTG and EVG also decreased the frequency and magnitude of intracellular calcium transients. Longer ARV exposures and simultaneous exposure to multiple ARVs increased the magnitude of these neurotoxic effects. Using the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased hNPC viability and changed the distribution of histone modifications that regulate chromatin packing, suggesting that TDF may reduce neuroprogenitor pools important for CNS development and maintenance of cognition in adults. This study establishes human preclinical assays that can screen potential ARVs for CNS toxicity to develop safer cART regimens and HAND therapeutics.

neuroscience↗