Genetic Mapping and Phenotypic Analysis of GstE14 E.4.1 on Eye and Antennae Development in Drosophila melanogaster.

Genetic screens are valuable for identifying novel genes involved in the regulation of developmental processes. To identify genes associated with cell growth regulation in Drosophila melanogaster , a mutagenesis screen was performed. Undergraduate students participating in Fly-CURE phenotypically characterized the E.4.1 mutant which is associated with rough eyes and antennae overgrowth. Following complementation analysis and subsequent genomic sequencing, E.4.1 was identified as a novel mutant allele of GstE14 , a gene involved in ecdysone biosynthesis important for the timing of developmental events. The abnormal eye and antenna phenotypes observed resulting from the loss of GstE14 suggest its role in tissue growth.

Fly-CURE, a multi-institutional CURE using Drosophila, increases students' confidence, sense of belonging, and persistence in research.

The Fly-CURE is a genetics-focused multi-institutional Course-Based Undergraduate Research Experience (CURE) that provides undergraduate students with hands-on research experiences within a course. Through the Fly-CURE, undergraduate students at diverse types of higher education institutions across the United States map and characterize novel mutants isolated from a genetic screen in Drosophila melanogaster. To date, more than 20 mutants have been studied across 20 institutions, and our scientific data have led to eleven publications with more than 500 students as authors. To evaluate the impact of the Fly-CURE experience on students, we developed and validated assessment tools to identify students' perceived research self-efficacy, sense of belonging in science, and intent to pursue additional research opportunities. Our data, collected over three academic years and involving 14 institutions and 480 students, show gains in these metrics after completion of the Fly-CURE across all student subgroups analyzed, including comparisons of gender, academic status, racial and ethnic groups, and parents' educational background. Importantly, our data also show differential gains in the areas of self-efficacy and interest in seeking additional research opportunities between Fly-CURE students with and without prior research experience, illustrating the positive impact of research exposure (dosage) on student outcomes. Altogether, our data indicate that the Fly-CURE experience has a significant impact on students' efficacy with research methods, sense of belonging to the scientific research community, and interest in pursuing additional research experiences.

Genetic mapping of the p47 L.3.2 mutation in Drosophilamelanogaster.

An EMS-based forward genetic screen was conducted in an apoptotic null background to identify genetic aberrations that contribute to regulation of cell growth in Drosophila melanogaster . The current work maps the genomic location of one of the identified mutants, L.3.2 . Genetic crosses conducted through the Fly-CURE consortium determined that the gene locus for the L.3.2 mutation is p47 on chromosome 2R.

Fly-CURE, a Multi-institutional CURE using Drosophila, Increases Students' Confidence, Sense of Belonging, and Persistence in Research.

The Fly-CURE is a genetics-focused multi-institutional Course-Based Undergraduate Research Experience (CURE) that provides undergraduate students with hands-on research experiences within a course. Through the Fly-CURE, undergraduate students at diverse types of higher education institutions across the United States map and characterize novel mutants isolated from a genetic screen in Drosophila melanogaster. To evaluate the impact of the Fly-CURE experience on students, we developed and validated assessment tools to identify students' perceived research self-efficacy, sense of belonging in science, and intent to pursue additional research opportunities. Our data show gains in these metrics after completion of the Fly-CURE across all student subgroups analyzed, including comparisons of gender, academic status, racial and ethnic groups, and parents' educational background. Importantly, our data also show differential gains in the areas of self-efficacy and interest in seeking additional research opportunities between Fly-CURE students with and without prior research experience, illustrating the positive impact of research exposure (dosage) on student outcomes. Altogether, our data indicate that the Fly-CURE experience has a significant impact on students' efficacy with research methods, sense of belonging to the scientific community, and interest in pursuing additional research experiences.

The I.3.2 developmental mutant has a single nucleotide deletion in the gene centromere identifier.

The mutation I.3.2 was previously identified in a FLP/FRT screen of chromosome 2R for conditional growth regulators. Here we report the phenotypic characterization and genetic mapping of I.3.2 by undergraduate students participating in Fly-CURE, a pedagogical program that teaches the science of genetics through a classroom research experience. We find that creation of I.3.2 cell clones in the developing eye-antennal imaginal disc causes a headless adult phenotype, suggestive of both autonomous and non-autonomous effects on cell growth or viability. We also identify the I.3.2 mutation as a loss-of-function allele of the gene centromere identifier ( cid ), which encodes centromere-specific histone H3 variant critical for chromosomal segregation.

Patched and Costal-2 mutations lead to differences in tissue overgrowth autonomy.

Genetic screens are used in Drosophila melanogaster to identify genes key in the regulation of organismal development and growth. These screens have defined signalling pathways necessary for tissue and organismal development, which are evolutionarily conserved across species, including Drosophila. Here, we have used an FLP/FRT mosaic system to screen for conditional regulators of cell growth and cell division in the Drosophila eye. The conditional nature of this screen utilizes a block in the apoptotic pathway to prohibit the mosaic mutant cells from dying via apoptosis. From this screen, we identified two different mutants that mapped to the Hedgehog signalling pathway. Previously, we described a novel Ptc mutation and here we add to the understanding of disrupting the Hh pathway with a novel allele of Cos2. Both of these Hh components are negative regulators of the pathway, yet they depict mutant differences in the type of overgrowth created. Ptc mutations lead to overgrowth consisting of almost entirely wild-type tissue (non-autonomous overgrowth), while the Cos2 mutation results in tissue that is overgrown in both the mutant and wild-type clones (both autonomous and non-autonomous). These differences in tissue overgrowth are consistent in the Drosophila eye and wing. The observed difference is correlated with different deregulation patterns of pMad, the downstream effector of DPP signalling. This finding provides insight into pathway-specific differences that help to better understand intricacies of developmental processes and human diseases that result from deregulated Hedgehog signalling, such as basal cell carcinoma.

Genetic Mapping of a new Hippo allele, HpoN.1.2, in Drosophila melanogaster.

Genetic screens provide a mechanism to identify genes involved with different cellular and organismal processes. Using a Flp/FRT screen in the Drosophila eye we identified mutations that result in alterations and de-regulation of cell growth and division. From this screen a group of undergraduate researchers part of the Fly-CURE consortium mapped and characterized a new allele of the gene Hippo, HpoN.1.2.

Chronology, magnitude and duration of expression of putative sex-determining/differentiation genes in a turtle with temperature-dependent sex determination.

The red-eared slider turtle (Trachemys scripta) possesses temperature-dependent sex determination (TSD) in which the incubation temperature determines gonadal sex. Although a number of mammalian gene homologues have been identified in reptiles with TSD, the exact sex-determining trigger(s) is not known. To date, the current study represents the most comprehensive simultaneous evaluation of the chronology of mRNA expression profiles of putative sex-determining/differentiation genes (Dmrt1, Sox9, Amh, Lhx9, and Foxl2) from gonads incubated at male- and female-producing temperatures in T. scripta. Additionally, sex-reversing treatments with 17ß-estradiol and letrozole were examined. At a male-producing temperature, Dmrt1 expression was sexually dimorphic by stage 17, Sox9 by 19 and Amh by 21. In contrast, Foxl2 did not significantly increase until after the thermosensitive period at a female-producing temperature. Treatment with 17ß-estradiol resulted in reduced gonad size and/or inhibited gonadal development and differentiation. Gene expression was subsequently low in this group. Sex reversal utilizing letrozole failed to produce testes at a female-producing temperature and as such, gene expression was comparable to ovary. These results indicate that Dmrt1 and Sox9 are potential triggers for testis differentiation and Amh, Lhx9 and Foxl2 represent a conserved core set of genes in the sex-determining/differentiation pathway of TSD species.

The cloning and expression analysis of Lhx9 during gonadal sex differentiation in the red-eared slider turtle, Trachemys scripta, a species with temperature-dependent sex determination.

Many reptiles, including the red-eared slider turtle (Trachemys scripta), possess a temperature-dependent sex determination (TSD) mechanism where the temperature at which the developing embryos are incubated dictates the gonadal sex of the animal. A number of mammalian gene orthologues have been identified in the sex determination/differentiation cascade of reptiles with TSD although the exact trigger(s) is not well understood. A potential early regulator of gonadal differentiation, Lhx9, controls the proliferation of gonadal cells in mice and its absence prevents gonadal development and drastically reduces the expression of Sf-1, a gene that regulates the expression of steroidogenic enzymes in the bipotential gonad. In the current study, we cloned Lhx9 from T. scripta and analyzed its expression throughout the thermosensitive period of gonad development using quantitative PCR. We examined the expression profiles of Lhx9 in embryos incubated under control conditions at male- and female-producing temperatures and with the application of exogenous 17ß-estradiol or an aromatase inhibitor, Letrozole, to induce sex reversal. The T. scripta Lhx9 cDNA and predicted amino acid sequence showed high homology to those of chicken, anole, and mouse. Lhx9 was expressed at both male- and female-producing temperatures with expression levels increasing throughout the thermosensitive period. Letrozole induced sex-reversal did not alter Lhx9 expression levels. 17ß-estradiol treatments appeared to inhibit or delay gonadal differentiation and resulted in lower Lhx9 expression from the presumptive gonadal ridge region. The structural homology and temporal expression pattern of Lhx9 suggests that this represents a conserved element in the gonadal differentiation cascade of T. scripta.