Browsing by Author "Lakdawala, Mohammed Farhan"
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Item Genetic interactions between the DBL-1/BMP-like pathway and dpy body size–associated genes in Caenorhabditis elegans(American Society for Cell Biology, 2019-12-12) Lakdawala, Mohammed Farhan; Madhu, Bhoomi; Faure, Lionel; Vora, Mehul; Padgett, Richard W.; Gumienny, Tina L.Bone morphogenetic protein (BMP) signaling pathways control many developmental and homeostatic processes, including cell size and extracellular matrix remodeling. An understanding of how this pathway itself is controlled remains incomplete. To identify novel regulators of BMP signaling, we performed a forward genetic screen in Caenorhabditis elegans for genes involved in body size regulation, a trait under the control of BMP member DBL-1. We isolated mutations that suppress the long phenotype of lon-2, a gene that encodes a negative regulator that sequesters DBL-1. This screen was effective because we isolated alleles of several core components of the DBL-1 pathway, demonstrating the efficacy of the screen. We found additional alleles of previously identified but uncloned body size genes. Our screen also identified widespread involvement of extracellular matrix proteins in DBL-1 regulation of body size. We characterized interactions between the DBL-1 pathway and extracellular matrix and other genes that affect body morphology. We discovered that loss of some of these genes affects the DBL-1 pathway, and we provide evidence that DBL-1 signaling affects many molecular and cellular processes associated with body size. We propose a model in which multiple body size factors are controlled by signaling through the DBL-1 pathway and by DBL-1–independent processes.Item Identifying and characterizing regulators of a bone morphogenetic protein cell signaling pathway in Caenorhabditis elegans(2022-12-01T06:00:00.000Z) Lakdawala, Mohammed Farhan; Gumienny, Tina L; Averitt, Dayna L; Brower, Christopher; Savage-Dunn, Cathy; Spencer, JulietCell-to-cell communication pathways ensure proper development of organisms. One group of signaling pathways that animals use is the bone morphogenetic protein (BMP) family. BMPs play significant roles in embryogenesis, development of skeletal and cardiovascular systems, among many others. Misregulation of BMP signaling leads to various diseases and thus needs to be tightly regulated. An understanding of how this pathway is itself controlled and performs diverse functions throughout the lifetime of living organisms is not very well understood. To identify new regulators of the BMP signaling pathway we used the roundworm C. elegans. DBL-1/BMP signaling is a dose dependent regulator of body size. Over expression of DBL-1 makes animal longer and suppression of the pathway reduces animal body size. Using genetic and imaging tools, we identified a group of genes called dumpy (dpy) that genetically interact with DBL-1/BMP signaling. Most of the identified dpy genes code for extracellular matrix components and/or are associated with body size regulation. We showed that loss of some dpy genes suppresses expression of the DBL-1 ligand and DBL-1 signaling activity. Using RNA sequencing, we identified genes that are differentially regulated by the DBL-1 signaling pathway; many of which regulate body size. Furthermore, we identified two dpy genes, dpy-2 and dpy-9, that have stage-specific interactions with the DBL-1 signaling pathway. Additionally, we characterized the interaction between DBL-1 pathway and dpy-24/blmp-1. BLMP-1 is a transcription regulator and controls some organismal traits that the DBL-1 pathway also affects. We showed DBL-1 signaling and BLMP-1 regulate each other. We also identified a physical interaction between BLMP-1 and the SMADs, downstream regulator of DBL-1 pathway using yeast-two hybrid system. Using bioinformatics and qPCR analysis, we showed that the interaction between SMADs and BLMP-1 is required to regulate some common downstream target genes. Lastly, we performed epistasis studies and identified that the DBL-1 signaling pathway and BLMP-1 work partially independently to regulate body size and work together to regulate brood size. Collectively, we identified additional regulators and interactors of the DBL-1 signaling pathway, and our findings provide insights about a new molecular mechanism by which DBL-1 pathway regulate certain organism traits.Item Loss of dpy-2 and dpy-9 has stage-specific effects on DBL-1 pathway signaling(Caltech Library, 2019-12) Lakdawala, Mohammed Farhan; Gumienny, Tina L.Loss of some cuticle collagens negatively affects DBL-1 pathway signaling in a stage-dependent manner (Lakdawala et al. 2019; Madaan et al. 2019). We previously observed that in one-day old adult animals, loss of dpy-2 or dpy-9 had no effect on GFP::DBL-1 expressed from the dbl-1 promoter (Beifuss and Gumienny 2012; Lakdawala et al. 2019). We also observed that expression of spp-9p::gfp, a reporter that is negatively regulated by the DBL-1 pathway, was not affected in one-day old adult animals (Roberts et al. 2010; Lakdawala et al. 2019). Post-embryonic expression of dpy-2 and dpy-9 is highest in L2 and L3, but low in L4 and even lower in young adults (Gerstein et al. 2010). Because cuticle secreted in one stage creates the cuticle in the next stage, this is consistent with the observation that loss of dpy-2 and dpy-9 has no effect on DBL-1 signaling in the adult (Hall and Altun 2008; Lakdawala et al. 2019). However, the DPY-2 and DPY-9 expression patterns led us to ask if DBL-1 signaling is affected at L4 by loss of dpy-2 or dpy-9. To our surprise, we found that dpy-2(e8) or dpy-9(e12) resulted in significant increases of GFP::DBL-1 fluorescence within DBL-1-secreting cells in L4 animals compared to control populations (Figure 1, Table 1). We also tested DBL-1 pathway reporter activity in these dpy-2 and dpy-9 mutants. Consistent with the increased GFP::DBL-1 fluorescence at L4, we observed significantly decreased fluorescence from the spp-9p::gfp reporter at L4 (Figure 1, Table 1). These results are consistent with DPY-2 and DPY-9 affecting DBL-1 signaling at the L4 stage but not at the adult stage. This suggests that these two collagens have a stage-specific effect on DBL-1 signaling, but this effect is normally inhibitory, as loss of dpy-2 or dpy-9 increased GFP::DBL-1 fluorescence and decreased spp-9p::GFP fluorescence.Item Small-scale extraction of Caenorhabditis elegans genomic DNA(JOVE, 2022) Madhu, Bhoomi; Lakdawala, Mohammed Farhan; Gumienny, Tina L.Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCRbased applications.Item The DBL-1/TGF-β signaling pathway tailors behavioral and molecular host responses to a variety of bacteria in Caenorhabditis elegans(2023-09-26) Madhu, Bhoomi; Lakdawala, Mohammed Farhan; Gumienny, Tina L.Generating specific, robust protective responses to different bacteria is vital for animal survival. Here, we address the role of transforming growth factor β (TGF-β) member DBL-1 in regulating signature host defense responses in Caenorhabditis elegans to human opportunistic Gram-negative and Gram-positive pathogens. Canonical DBL-1 signaling is required to suppress avoidance behavior in response to Gram-negative, but not Gram-positive bacteria. We propose that in the absence of DBL-1, animals perceive some bacteria as more harmful. Animals activate DBL-1 pathway activity in response to Gram-negative bacteria and strongly repress it in response to select Gram-positive bacteria, demonstrating bacteria-responsive regulation of DBL-1 signaling. DBL-1 signaling differentially regulates expression of target innate immunity genes depending on the bacterial exposure. These findings highlight a central role for TGF-β in tailoring a suite of bacteria-specific host defenses.