From Nervous System Changes to Systemic Change in Science: Investigating Sex Differences in the Impact of Maternal Immune Activation on Offspring Brain Development and Behavior, and Building Social Justice in Science Through Pedagogy and Community Initiatives to Increase Belonging

Laboratory research on sex differences in response to maternal immune activation:/nWe investigated sex differences in molecular and behavioral responses to maternal immune activation during pregnancy. Epidemiological evidence indicates that immune activation during pregnancy impacts brain development, leading to increased incidence of neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. Researchers have modeled this association in the laboratory using maternal immune activation (MIA) animal models, in which offspring exposed to MIA in development demonstrate changes in social and restricted repetitive behavior that recapitulate aspects of autistic behaviors in humans. My research seeks to understand the nature of and mechanisms underlying sex differences in the fetal response to maternal immune activation. Male offspring generally demonstrate greater magnitude and/or number of molecular and behavioral changes in response to immune activation during gestation compared to females. Sex differences in the MIA model are understudied, and little research has considered sex as a biological variable with the potential to impact immune response mechanisms in the MIA context./nWe first used a hypothesis-driven approach to investigate the role of sex hormone pathways in macrophages as a potential sex-specific mechanism regulating the immune response in the fetal brain. The estrogen receptor β (ERβ) has been shown to negatively regulate transcription of pro-inflammatory immune response genes in microglia. We hypothesized that female and male embryos may differ in the level of ERβ signalling in brain macrophages, leading to differential immune responses and therefore the observed sex differences in neurodevelopment after maternal immune activation. To test this hypothesis, we used the Cre-Lox system to generate mouse offspring with a knockdown of estrogen receptor β (ERβ) expression in myeloid cells using cre recombinase driven by the Cx3cr1 promoter (Cre+). We then compared the behavior of Cre+ and Cre- offspring, with or without maternal immune activation via polyinosinic–polycytidylic acid (Poly(I:C)) at embryonic day 12.5. We observed the adult behavior of 94 offspring from 6 vehicle-treated and 9 Poly(I:C)-treated litters, using 11 assays measuring social, restricted repetitive, anxiety-like, depression-like, and motor behaviors. Overall we found few significant differences between experimental groups in this study, potentially due to a low 2mg/kg dose of Poly(I:C), reduced statistical power because of unexpectedly low sample sizes for control groups, and sources of variability specific to the MIA model such as maternal immune responsiveness. However, we did observe a few significant behavior differences, including: 1) baseline sex differences in marble burying with increased number of buried marbles in males, 2) female-specific effects of Cre genotype and Poly(I:C) treatment in the tail suspension test (TST), a measure of depression-like behavior, and 3) a decrease in distance traveled in the elevated plus maze in Poly(I:C)-treated animals. These findings suggest potential roles for sex in modulating restricted repetitive behavior and depression-like behavior, for macrophage ERβ signaling in regulating depression-like behavior in females, and for Poly(I:C) maternal immune activation in altering depression- and anxiety-like behavior. However, the lack of expected Poly(I:C)-induced changes in social and restricted repetitive behavior made it difficult to evaluate our hypothesis about the role of macrophage ERβ in sex differences after MIA./nWe next undertook an exploratory single-cell RNA sequencing study to better understand sex differences in molecular pathways in the fetal brain in response to maternal immune activation. We stimulated pregnant female mice with 10mg/kg Poly(I:C) at E12.5 based on a dosing study that found sickness phenotypes but not litter loss at this dose. We isolated fetal brains 6 or 24 hours after injection, pooling equal numbers of fetal female and male brains at 6 hours and preparing separate female and male samples at 24 hours. We enriched for myeloid cells and prepared single cell RNA sequencing libraries using a fixation and plate-based protocol. We sequenced 975 million 150bp paired-end reads, resulting in 18465 cells and 16091 features in the final data set after filtering. Clustering resulted in 13 clusters, and cell types were identified using marker gene expression as well as comparison to reference data sets. Cell types included ependymal cells, myeloid cells, neural cells, and endothelial cells. Differential gene expression analysis between experimental groups within cell type clusters found 62 significantly differentially expressed genes (DEGs), with the majority observed in ependymal cells. Further examining the DEGs using gene set analysis, we observed 66 significant gene ontology terms for the set of 11 genes upregulated in female ependymal cells 24hr after injection with Poly(I:C) compared to vehicle, primarily relating to regulation of the immune response. We also found significant gene ontology terms relating to cilia, cell transport, and motility when examining the genes differentially expressed in male neural progenitor cells/radial glia 24hr after injection with Poly(I:C) compared to vehicle. These findings represent the first evidence to our knowledge that ependymal cells in the fetal brain may be responding to maternal immune activation. In addition, sex differences in fetal ependymal cells have not been previously reported. The observed transcriptional immune response in female ependymal cells and upregulation of cilia genes in male neural progenitor cells 24hr after Poly(I:C) treatment add to our understanding of fetal responses to MIA as well as sex differences in early brain development./nEvidence-based teaching and learning with the MCB Distance Learning Task Force:/nIn Spring 2020, instruction and learning at UC Berkeley largely shifted from in-person to online due to the COVID-19 pandemic. To assist this transition within the Molecular and Cell Biology (MCB) Department and promote effective and equitable pedagogy, I founded and led the MCB Distance Learning Task Force. Collectively we designed and analyzed an undergraduate survey on the remote learning experience, and published a website for instructors with recommendations on teaching remotely based on the education literature. In our survey, administered in July 2020, 134 students reported their experience on a breadth of issues, including: 1) an overall negative impact of remote instruction on their learning, 2) their preferences for specific learning formats and technologies. 3) barriers to attending synchronous classes, 4) generally reliable access to technology and internet, 5) service needs, 6) understanding of and preferences for grading policies, and 7) concerns about cheating and equity on exams. These findings expand our understanding of MCB students' experiences and preferences about remote learning during the initial phase of the COVID-19 pandemic. Our literature review of best practices in STEM and online education was summarized in four main recommendations to instructors and published as a website with supporting information. Our main recommendations included: 1) Create an inclusive online learning environment, 2) Provide clear course materials organized within a structured website, 3) Help students connect with each other and instructors, and 4) Develop effective and fair assessments. Combining information gathered from our survey as well as our literature review, we compiled recommendations for MCB departmental leadership on actions to take to improve the remote learning and teaching experience, as well as hosted 2 training sessions for faculty and graduate student instructors on best practices. The information and recommendations the Task Force gathered apply beyond the specific context of the COVID-19 pandemic, and we hope the resources we developed will continue to be used during in-person learning and to weather future crises. Improving classroom culture, increasing student engagement, communicating about course and department policies, updating assessment structures, and providing services to students in need will always be critical to teaching and learning effectively./nProgram evaluation with the Inclusive MCB initiative:/nSystems of oppression within science and academia have led to discrimination of under-represented minority (URM) and other marginalized scientists. The Inclusive MCB (iMCB) initiative was formed to create capacity within the MCB department at UC Berkeley to discuss and address issues affecting URM scientists. Among the programs initiated by iMCB include annual conferences presenting scholarly research about improving scientific culture and practices in pursuit of equity, highlighting the experiences of URM scientists, and providing opportunities to build community. The impact of the conferences were assessed by the iMCB Assessment Team using pre- and post-conference surveys. Here, I present findings from our assessment of the Fall 2020 "Building a Sense of Belonging" iMCB Conference. We found that the keynote talks and affinity groups were the most impactful components of the conference program, and that keynotes were rated more impactful by URM participants than non-URM participants. Attendees also had a stronger sense of belonging to the community of scientists and an increased comfort sharing about their values and identities after the conference. Sense of belonging increased for both URM and non-URM groups, and gaps in sense of belonging were observed both before and after the conference, with non-URMs having a higher sense of belonging in both samples. We found that respondents planned to be involved in justice, equity, diversity, and inclusion work in the coming year to a greater extent after the conference. Our results showed a clear positive impact of the conference for both URM and non-URM participants, leading to increased personal sense of belonging as well as sentiments like comfort sharing values and plans to be involved that have the potential to impact interpersonal and organizational culture. Our results also demonstrate the continued inequities in science, with non-URM participants having a higher sense of belonging than URM participants overall. These findings underscore the need to continue improving culture and policies in science and point toward potential avenues for organizing. Indeed, these results inspired organizers to create an 8-week affinity groups program in summer 2021 and continue hosting keynotes and affinity groups at future annual conferences.

Advisor: Saijo, Kaoru