63 Modeling Microcephaly in Drosophila

Faculty Mentor: Nichole Link (Neurobiology, University of Utah)

Abstract

Microcephaly is a neurodevelopmental disorder characterized by significantly reduced occipital frontal circumference (OFC). It can be accompanied by comorbidities including intellectual disabilities, seizures, and other developmental phenotypes. Microcephaly is attributed to malnutrition and exposure to toxins or infection during pregnancy, but genetic mutations are also a leading cause. To investigate the genetic mechanisms behind microcephaly, our lab collaborates with a physician who has completed whole exome sequencing for two hundred microcephaly patients. He identified potentially pathogenic human variant genes, which we are studying using Drosophila melanogaster as our model organism. We screened for loss of function phenotypes through RNAi knockdown of fly orthologs to determine if these genes are necessary for brain development. In our primary screen, we knocked down the genes in neural stem cells or mature neurons and assessed third-instar larval brain volume.

Introduction

Microcephaly is a neurodevelopmental disorder that results in a reduced occipital frontal circumference more than 2 standard deviations below the mean. The disorder can be accompanied by several comorbidities, including seizures, learning disabilities, and other developmental conditions [1]. Microcephaly affects individuals for their entire life, and there is currently no cure [1]. However, developing an understanding of the disorder and its causes can provide emotional relief for patients and family members. Microcephaly is attributed to viral infections and toxin exposure during pregnancy, as well as genetic mutations [1]. As of 2023, 30 genes have been identified to be associated with severe primary microcephaly, but this list is not exhaustive [2, 3]. We utilize model organisms to study candidate genes from human patients in order to discover genetic causes of microcephaly.

Drosophila melanogaster are a beneficial model organism for genetic studies, as many human genes have Drosophila orthologs with genetic function conserved between species [4, 5]. Additionally, the short generation time and high numbers of progeny make Drosophila an excellent model organism to study genetic effects on neurodevelopmental disorders. These organisms are also highly receptive to genetic manipulation, and the UAS/GAL4 system allows for the expression of different target genes of interest in a tissue-specific manner. We used the UAS/GAL4 system to express RNAi and knockdown candidate genes in either post-mitotic neurons or neural stem cells [5]. Previous studies investigating microcephaly-related genes have demonstrated further abnormal phenotypes including reduced brain volume, increased apoptosis, and decreased proliferation [6]. In this study, we used brain lobe volume as a measure of neurodevelopment and microcephalic phenotypes.

Methods

To determine if a candidate gene was necessary for neurodevelopment, we used the GAL4 drivers inscuteable (insc) and neuronal synaptobrevin (nsyb) to knockdown each gene in neural stem cells and post-mitotic neurons respectively. [4, 7]. Virgin female flies with the GAL4 driver were crossed with adult male Drosophila with the UAS-RNAi line of interest. These crosses were set at 29 degrees Celsius on blue glucose food. We then identified the late third-instar larval stage by anatomy (spiracles) and intestinal clearance of blue food. To measure brain lobe volume, we dissected third instar larval brains and stained them for Deadpan (neural stem cells) and DAPI (nuclei). We then used confocal microscopy with the 40X water immersion lens to image one lobe from each brain. Z-stacks size was set at 2 µm and set to include the full brain lobe. We performed brain volume analysis using IMARIS software to trace the brain at every 5th z stack and compile these surfaces together to automatically generate volume. We analyzed 74 RNAi lines under insc driver and 79 RNAi lines under nsyb driver in cohorts of 3 to 4 experimental RNAi lines and an EGFP RNAi control. Kruskal- Wallis tests were performed to compare all genotypes against pooled controls using GraphPad Prism [4, 7].

Results

We compared the volumes of each RNAi knockdown group to the EGFP knockdown control using the Kruskal-Wallis test due to unequal variances to determine if knockdown of candidate genes resulted in a change in brain lobe volume. Under the insc-GAL4 driver, RNAi knockdown of 9 out of 42 genes resulted in brain volume that differed significantly from the control (Figure 1). Figure 2 shows brightfield images comparing brain lobe volume of EGFP (control) and two candidate genes (dom and Ran). These genes exhibit significantly reduced brain lobe volume with RNAi knockdown in neural stem cells. Similarly, under the nsyb-GAL4 driver, we found that RNAi knockdown of 10 out of 42 candidate genes resulted in brain volume that differed significantly from our control (Figure

3). Figure 4 shows brightfield images comparing RNAi knockdown of EGFP control against Bx42 and Ack in mature neurons. RNAi knockdown of Bx42 and Ack under the nsyb-GAL4 driver resulted in significantly reduced brain lobe volume.

Figure 1. RNAi knockdown under the insc-GAL4 driver resulted in significantly different brain lobe volume for 9 out of 42 genes (74 RNAi lines). EGFP RNAi knockdown control shown in green and significantly different candidate gene RNAi knockdown shown in pink.

Figure 2. Brightfield images of third-instar larval brains with RNAi knockdown of dom and Ran in neural stem cells resulted in significantly smaller brain lobe volume.

Figure 3. RNAi knockdown under the nsyb-GAL4 driver resulted in significiantly smaller brain lobe volume for 10 out of 42 genes (79 RNAi lines). EGFP RNAi knockdown control shown in green and significantly different candidate gene RNAi knockdown shown in pink.

Figure 4. Brightfield images of third-instar larval brains with RNAi knockdown of Bx42 and Ack in mature neurons resulted in significantly smaller brain lobe volume.

Discussion/Conclusion

We found that knockdown in either neural stem cells or neurons resulted in significantly different brain volume in approximately 21 or 24 percent of our genes under the neural stem cell driver or mature neuron driver, respectively. This relatively high hit rate is reasonable, as candidate genes were implicated in neurodevelopment through patient data. The results of the screen indicate that these fly genes may be involved in neurodevelopmental pathways, and prompt further investigation into the human orthologs. Although there are many potential molecular and cellular mechanisms for reduced brain lobe volume, we hypothesize decreased stem cell proliferation and increased apoptosis to be likely causes. Specifically, we predict that RNAi knockdown in neural stem cells may result in decreased proliferation and decreased brain lobe volume. Likewise, RNAi knockdown in post-mitotic neurons may cause increased apoptosis and decreased brain lobe volume. Our next step will be following up this screen with a secondary screen to investigate cellular phenotypes in significantly small brains. We will determine if apoptosis and proliferation are affected by knockdown of candidate genes, and how this may contribute to reduced brain lobe volume. Furthermore, we will investigate the role of human orthologs in neurodevelopment through rescue experiments. We anticipate that functionally conserved wild type human orthologs will rescue neurodevelopmental phenotypes, while human variants will not rescue and may be implicated as pathogenic.

Acknowledgements

University of Utah, Physician Collaborator Dr. Xiao Mao for candidate genes

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. (2139322). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

This work was supported by UROP from the Office of Undergraduate Research at the University of Utah awarded to Adriana Bibo.