Gene expression and data analysis pipeline using cancer BioPortal in the classroom

At institutions with an emphasis on authentic research experiences as an integral part of the biology curriculum, COVID created a huge challenge for course instructors whose learning objectives were designed for such experiences. Moving such laboratory experiences online when remote learning became necessary has resulted in a new model for CUREs that utilizes free online databases to provide not only a novel research experience for students, but also the opportunity to engage in big data analysis. Cancer BioPortal (cBioPortal) is an open-access collective cancer research resource for storing and exploring clinical, genomic, proteomic, and transcriptomic data. cBioPortal eliminates the computational barrier of interpreting complex genomic data by providing easily understandable visualization that can be interpreted and translated into relevant biological insights. Because no prior computational knowledge is required, cBioPortal is an ideal educational tool for either in-person or distance learning environments. We developed a pedagogical approach, video tutorials, and data analysis workflows centered on using cBioPortal. Pedagogically, students develop an initial research outline that is continually updated and graded throughout the project. Progress during the project or course is assessed by a series of student presentations that are 5 to 15 minutes in length and are aimed at explaining the approach used in data acquisition, interpretation of the data, and relevance to the initial hypothesis. While cancer-specific, this analysis platform appeals to a wide range of classes and student interests. Further, the project has been successfully done both as an independent research experience and as part of a virtual class-based research project

Expression Profiling of MAP Kinase–Mediated Meiotic Progression in Caenorhabditis elegans

The LET-60 (Ras)/LIN-45 (Raf)/MPK-1 (MAP kinase) signaling pathway plays a key role in the development of multiple tissues in Caenorhabditis elegans. For the most part, the identities of the downstream genes that act as the ultimate effectors of MPK-1 signaling have remained elusive. A unique allele of mpk-1, ga111, displays a reversible, temperature-sensitive, tissue-specific defect in progression through meiotic prophase I. We performed gene expression profiling on mpk-1(ga111) animals to identify candidate downstream effectors of MPK-1 signaling in the germ line. This analysis delineated a cohort of genes whose expression requires MPK-1 signaling in germ cells in the pachytene stage of meiosis I. RNA in situ hybridization analysis shows that these genes are expressed in the germ line in an MPK-1-dependent manner and have a spatial expression pattern consistent with the location of activated MPK-1. We found that one MPK-1 signaling-responsive gene encoding a C(2)H(2) zinc finger protein plays a role in meiotic chromosome segregation downstream of MPK-1. Additionally, discovery of genes responsive to MPK-1 signaling permitted us to order MPK-1 signaling relative to several events occurring in pachytene, including EFL-1/DPL-1 gene regulation and X chromosome reactivation. This study highlights the utility of applying global gene expression methods to investigate genes downstream of commonly used signaling pathways in vivo

MEG-1 and MEG-2 Are Embryo-Specific P-Granule Components Required for Germline Development in Caenorhabditis elegans

In Caenorhabditis elegans, germ granules called P granules are directly inherited from mother to daughter and segregate with the germ lineage as it separates from the soma during initial embryonic cell divisions. Here we define meg-1 and meg-2 (maternal-effect germ-cell defective), which are expressed in the maternal germline and encode proteins that localize exclusively to P granules during embryonic germline segregation. Localization of MEG-1 to P granules depends upon the membrane-bound protein MES-1. meg-1 mutants exhibit multiple germline defects: P-granule mis-segregation in embryos, underproliferation and aberrant P-granule morphology in larval germ cells, and ultimately, sterility as adults. The penetrance of meg-1 phenotypes increases when meg-2 is also absent. Loss of the P-granule component pgl-1 in meg-1 mutants increases germ-cell proliferation, while loss of glh-1 decreases proliferation. Because meg-1 is provided maternally but its action is required in the embryonic germ lineage during segregation from somatic lineages, it provides a critical link for ensuring the continuity of germline development from one generation to the next

Meiotic Progression and MAP Kinase Activation Is Restored after Shift to Permissive Temperature

<div><p>Dissected gonads were stained with DAPI (left column) and α-diP MAP kinase (middle column). Boxed area in left column is enlarged in right column (inset) to show germ cell morphology during meiotic progression. Asterisks indicate distal end of germ line. Arrowheads indicate nuclei in diakinesis. Brackets indicate region with activated MAP kinase staining.</p><p>(A) Gonads from control animals.</p><p>(B) <i>mpk-1</i> gonads at 26 °C (0 h) and following shift to 15 °C (3–12 h).</p><p>All gonads also carry <i>fem-1(hc17)</i> and <i>unc-79(e1068)</i> alleles. Scale bar, 20 μm.</p></div

MPK-1 and EFL-1/DPL-1 Do Not Regulate the Same Set of Genes in the Germ Line

<p>MPK-1 signaling-responsive genes identified in this study have minimal overlap with candidate down-regulated or up-regulated EFL-1/DPL-1-responsive genes (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020174#pgen-0020174-st001" target="_blank">Table S1</a>).</p

Candidate MPK-1 Signaling-Responsive Autosomal Genes Depend on <i>mpk-1</i> for Expression

<p>RNA in situ hybridizations of N2 (A, B, D, E, G, and H) and <i>mpk-1</i> (C, F, and I) gonads. Sense and AS probes correspond to three MPK-1 signaling-responsive genes on autosomes: <i>pzf-1</i> (A–C), <i>sea-1/tbx-18</i> (D–F), and <i>hlh-2</i> (G–I). Only medial and proximal arm of gonads are shown; no staining was detected in the distal gonad for any probe. Arrow indicates location of late pachytene region. Scale bar, 20 μm.</p

Expression of E2F-Regulated Genes Does Not Require MPK-1 Signaling

<div><p>(A) In situ hybridization of N2, <i>mpk-1,</i> and <i>dpl-1</i> gonads stained with <i>rme-2</i> and <i>cpg-3</i> AS probes. Sense probes showed no signal.</p><p>(B) N2 and <i>mpk-1</i> gonads stained with an antibody to the RME-2 protein (red) and DAPI to mark nuclei (blue).</p><p>Scale bar, 20 μm.</p></div

<i>pzf-1</i> Displays a Reduced Brood Size in the <i>mpk-1</i> Background

<div><p>(A) Schematic of <i>pzf-1</i> genomic locus showing location of <i>vr3</i> deletion (top) and predicted PZF-1 protein (bottom), with predicted C₂H₂ zinc finger domains in black. Numbers indicate amino acid residue of start of each C₂H₂ zinc finger pair.</p><p>(B) Chart of mean live progeny per animal of N2, <i>pzf-1(vr3), unc-79(e1068)</i>, <i>unc-79(e1068)mpk-1(ga111),</i> and <i>unc-79(e1068)mpk-1(ga111);pzf-1(vr3)</i> at 20 °C, 23 °C, and 25 °C. The total number of progeny from six to 24 hermaphrodites was counted for each temperature. Error bars indicate standard deviation.</p><p>(C) Ovulations/gonad arm/hour of N2, <i>pzf-1(vr3), unc-79(e1068)mpk-1(ga111),</i> and <i>unc-79(e1068)mpk-1(ga111);pzf-1(vr3).</i> The <i>unc-79(e1068)mpk-1(ga111)</i> strain showed a statistically significant difference from <i>unc-79(e1068)mpk-1(ga111);pzf-1(vr3)</i>. Because ovulation rates were determined on groups of N2 and <i>pzf-1(vr3)</i> worms, standard deviation was not calculated.</p></div

MPK-1 Signaling in the Germ Line

<div><p>(A) Model of multiple MPK-1 signaling-dependent effectors acting together to promote meiotic progression. X and Y represent putative proteins that function downstream of MPK-1. Additional processes besides chromosome segregation that contribute to meiotic progression are indicated by the question mark.</p><p>(B) Representation of events in germ line development. MPK-1 signaling occurs downstream of EFL-1/DPL-1 gene regulation, but prior to physiological germ cell death, X chromosome transcriptional activation, and GLD-1 down-regulation.</p></div

Temporal Expression Profiling of Meiotic Progression

<div><p>(A) Adults were shifted to permissive temperature (47 h after hatching) and subsequently analyzed at indicated times.</p><p>(B) Hierarchical clustering analysis of expression profiles upon resumption of MPK-1 signaling. Each column represents the mean of three replicates for each time sampled. Each row represents one of 90 genes with enriched expression in control compared with <i>mpk-1</i> at 0 h. Low expression in <i>mpk-1</i> relative to the control (black) is evident at 0 h, with increasing expression as meiotic progression resumes (yellow). Three major clusters, each with an expression correlation > 0.95, are indicated (strong, medium, and weak).</p></div