A functional genomics screen identifying blood cell development genes in Drosophila by undergraduates participating in a course-based research experience

Abstract

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Keywords: CURE; RNAi; blood; education; hematopoiesis.

Figure 1

A functional genomics screen for new hematopoietic genes in Drosophila. In the primary screen, RNAi occurred throughout the larval hematopoietic system, which specifically expressed GFP. Briefly, HHLT-GAL4 UAS-GFP flies (Mondal et al. 2014) were crossed to flies carrying different UAS-hairpin RNA (hpRNA) transgenes targeting a unique gene. Progeny third-instar larvae expressed both hpRNAs (eliciting an RNAi response) and GFP throughout the blood system. Expression of GFP was monitored by fluorescence microscopy in whole larvae, four at a time. RNAi lines causing a discernable increase or decrease in GFP fluorescence, relative to control larvae lacking RNAi, were selected for use in the secondary screen. In the secondary screen, RNAi line “hits” from the primary screen were crossed to population-specific GAL4 driver lines (Hml^Δ-GAL4 for maturing cells, dome-GAL4 for progenitor cells, and Antp-GAL4 for Posterior Signaling Center cells). These GAL4 driver lines also carried Hml^Δ-DsRed as a reporter of blood cell maturation. Expression of DsRed was monitored by fluorescence microscopy in whole larvae, four at a time.

Figure 2

Select examples of candidate hematopoietic genes identified in the primary screen by HHLT-GAL4 UAS-GFP expression. For each image, four GFP-expressing, third-instar larvae are shown with anterior at the top and dorsal facing up. (A) control larvae lacking RNAi. (B)–(H) candidate genes that cause increased GFP fluorescence with RNAi. (I)–(P) candidate genes that cause decreased GFP fluorescence with RNAi. Note that in panel O, arrows point to larvae affected by RNAi; the unaffected sibling larvae arise because of heterozygosity of the UAS-hpRNA transgene in the parental line. (Q)–(T) candidate genes altering GFP expression in the circulating cell population. Target genes and specific RNAi lines are indicated in the lower left of the panel. Black spots observable in some larvae (e.g., panel H) are melanotic pseudotumors. RNAi line designations: v, VDRC; t, TRiP.

Figure 3

Select examples of candidate hematopoietic genes identified in the secondary screen by Antp-GAL4 Hml^Δ-DsRed expression. For each image, four DsRed-expressing, third-instar larvae are shown with anterior at the top and dorsal facing up. (A) control larvae lacking RNAi. (B–H) candidate genes that cause increased DsRed fluorescence with RNAi. (I–P) candidate genes that cause decreased DsRed fluorescence with RNAi. Target genes and specific RNAi lines are indicated in the lower left of the panel. RNAi line designations: v, VDRC; t, TRiP; n, NIG.

Figure 4

Select examples of candidate hematopoietic genes identified in the secondary screen by dome-GAL4 Hml^Δ-DsRed expression. For each image, four DsRed-expressing, third-instar larvae are shown with anterior at the top and dorsal facing up. (A) control larvae lacking RNAi. (B–H) candidate genes that cause increased DsRed fluorescence with RNAi. (I–P) candidate genes that cause decreased DsRed fluorescence with RNAi. Target genes and specific RNAi lines are indicated in the lower left of the panel. RNAi line designations: v, VDRC; t, TRiP.

Figure 5

Select examples of candidate hematopoietic genes identified in the secondary screen by Hml^Δ-GAL4 Hml^Δ-DsRed expression. For each image, four DsRed-expressing, third-instar larvae are shown with anterior at the top and dorsal facing up. (A) control larvae lacking RNAi. (B–H) candidate genes that cause increased DsRed fluorescence with RNAi. (I–P) candidate genes that cause decreased DsRed fluorescence with RNAi. Target genes and specific RNAi lines are indicated in the lower left of the panel. RNAi line designations: v, VDRC; t, TRiP.

Figure 6

Impact of the URCFG experience on learning gains. (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 who responded to the survey (n = 265) reported increased gains across 20 of 21 different areas compared to students in the other groups. Scale: 1 = little to no gain, 2 = small gain, 3 = moderate gain, 4 = large gain, and 5 = very large gain. Error bars represent two times the standard error, representing greater than a 95% confidence interval. (B) average responses of URCFG students (green bars, top), when asked if they agreed with each of the statements on the left, regarding the impact of the course on their interest in science, ability to learn the process of scientific research and ability to learn the subject matter. Students scored each statement on a 5-point Likert scale, where 1 is “strongly disagree” and 5 is “strongly agree.” Scores are compared to those from students nationally in biology courses with a research component (red bars, bottom). See Materials and Methods for additional details.