Expression-Based Cell Lineage Analysis in Drosophila Through a Course-Based Research Experience for Early Undergraduates

Abstract

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Keywords: CURE; G-TRACE; STEM; education; gene expression.

Figure 1

Overview of the G-TRACE screening strategy. Transgenic Drosophila lines expressing GAL4 (enhancer-GAL4 lines; P{GawB} NP lines) are crossed to the G-TRACE screening stock. Progeny larvae will express GAL4 protein in various tissues, dependent upon enhancer activity, which will be reported by the expression of RFP (DsRed.T4). The GAL4-expressing cell will also initiate the cell lineage marker GFP, which will be expressed perpetually by all subsequent daughter cells (see Evans et al., 2009 for a complete description of the G-TRACE labeling mechanism). Wandering third-instar larvae from such crosses are collected, followed by the dissection of the brain, eye and wing imaginal discs, and the lymph gland (the hematopoietic organ). These tissues are subsequently mounted on glass slides for imaging by fluorescence microscopy, followed by analysis of RFP and GFP expression patterns. Using basic bioinformatics approaches, endogenous genes proximal to the GAL4 insertion site are identified. For each GAL4 line, representative fluorescence microscopy images, RFP/GFP expression data, and associated candidate regulatory gene information are assembled into the G-TRACE Expression Database (GED), a searchable, online database.

Figure 2

Incidence of GAL4 activity within the larval brain, eye and wing imaginal discs, and lymph gland. Bar graphs demonstrating the total number of GAL4-expressing lines identified per tissue (number above the bar, out of 563 screened) as well as the subset exhibiting either combined RFP and GFP expression (yellow) or GFP expression alone (green).

Figure 3

Select GAL4-expressing lines with complex G-TRACE patterns in the brain. A) Schematic of the third instar larval brain showing the primary structures identified during screening. B-L) Fluorescence microscopy images showing various patterns of real-time GAL4 activity (RFP, red) and associated cell lineages (GFP, green) within the third instar larval brain. The corresponding NP line identifier is shown in the upper right corner of each image. For all images, DNA is shown in blue (DAPI staining). Surface glia (SG); mushroom body (MB); central brain (CB); optic lobe (OL); medulla primordia (MP); lobula primordia (LOP); lamina primordia (LAP).

Figure 4

Select GAL4-expressing lines with complex G-TRACE patterns in the eye disc. A) Schematic of the third instar larval eye disc showing the primary structures identified during screening. B-I) Fluorescence microscopy images showing various patterns of real-time GAL4 activity (RFP, red) and associated cell lineages (GFP, green) within the third instar larval eye disc. The corresponding NP line identifier is shown in the upper right corner of each image. For all images, DNA is shown in blue (DAPI staining). Eye glia (EG); photoreceptors (PR); arista (AR); peripodial membrane (PM).

Figure 5

Select GAL4-expressing lines with complex G-TRACE patterns in the wing disc. A) Schematic of the third instar larval wing disc showing the primary structures identified during screening. B-I) Fluorescence microscopy images showing various patterns of real-time GAL4 activity (RFP, red) and associated cell lineages (GFP, green) within the third instar larval wing disc. The corresponding NP line identifier is shown in the upper right corner of each image. For all images, DNA is shown in blue (DAPI staining). Dorsal/ventral boundary (D/V); anterior/posterior boundary (A/P); peripodial membrane (PM).

Figure 6

Select GAL4-expressing lines with complex G-TRACE patterns in the lymph gland. A) Schematic of the third instar larval lymph gland showing the primary structures identified during screening. B-F) Fluorescence microscopy images showing various patterns of real-time GAL4 activity (RFP, red) and associated cell lineages (GFP, green) within the third instar larval lymph gland. The corresponding NP line identifier is shown in the bottom left corner of each image. For all images, DNA is shown in blue (DAPI staining). Primary lobes (PL); secondary and tertiary lobes (SL, TL); pericardial cell (PC); Cortical Zone (CZ); dorsal vessel (DV); posterior signaling center (PSC).

Figure 7

Impact of the URCFG experience on learning gains and STEM retention. A) Categorical data plot comparing reported learning gains between URCFG students (green triangles), students, nationally, completing summer research apprenticeships (All summer research students; blue diamonds), and students, nationally, completing introductory to advanced biology courses containing some research component (All students; red squares). Students participating in the URCFG exhibited increased gains across 21 different areas compared to students in the other groups. Learning gains were assessed using the Survey of Undergraduate Research Experiences (SURE) II, which offers both the Classroom Undergraduate Research Experiences (CURE) survey and the Summer Undergraduate Research Experience (SURE) survey. The CURE and SURE surveys include identical items that permit comparisons; URCFG students and “All students” took the CURE survey, while “All summer research students” took the SURE survey. The typical student in SURE cohorts was a third- or fourth-year student, and we compared to SURE 2013. Scale: 1 = little to no gain; 2 = small gain; 3 = moderate gain; 4 = large gain; 5 = very large gain. Error bars represent two times the standard error, representing greater than a 95% confidence interval. B) STEM retention rates are higher among URCFG students compared to national and UCLA averages. Degree completion data (6-year) is based on students enrolled in our URCFG CURE course from Winter 2003 through Spring 2018 (overall, n = 626; URM, n = 46). UCLA data were obtained from the Office of Analysis and Information Management (overall, n = 8,388; URM, n = 1,312). National data were obtained from Hurtado et al. (2012) (overall, n = 56,499; URM, n = 9,718). URCFG, Undergraduate Research Consortium for Functional Genomics; STEM, Science, Technology, Engineering, and Mathematics; URM, underrepresented minority.